Autotaxin interacts with lipoprotein(a) and oxidized phospholipids in predicting the risk of calcific aortic valve stenosis in patients with coronary artery disease.

BACKGROUND: Studies have shown that lipoprotein(a) [Lp(a)], an important carrier of oxidized phospholipids, is causally related to calcific aortic valve stenosis (CAVS). Recently, we found that Lp(a) mediates the development of CAVS through autotaxin (ATX). OBJECTIVE: To determine the predictive value of circulating ATX mass and activity for CAVS. METHODS: We performed a case-control study in 300 patients with coronary artery disease (CAD). Patients with CAVS plus CAD (cases, n = 150) were age- and gender-matched (1 : 1) to patients with CAD without aortic valve disease (controls, n = 150). ATX mass and enzymatic activity and levels of Lp(a) and oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB) were determined in fasting plasma samples. RESULTS: Compared to patients with CAD alone, ATX mass (P < 0.0001), ATX activity (P = 0.05), Lp(a) (P = 0.003) and OxPL-apoB (P < 0.0001) levels were elevated in those with CAVS. After adjustment, we found that ATX mass (OR 1.06, 95% CI 1.03-1.10 per 10 ng mL-1 , P = 0.001) and ATX activity (OR 1.57, 95% CI 1.14-2.17 per 10 RFU min-1 , P = 0.005) were independently associated with CAVS. ATX activity interacted with Lp(a) (P = 0.004) and OxPL-apoB (P = 0.001) on CAVS risk. After adjustment, compared to patients with low ATX activity (dichotomized at the median value) and low Lp(a) (<50 mg dL-1 ) or OxPL-apoB (<2.02 nmol L-1 , median) levels (referent), patients with both higher ATX activity (≥84 RFU min-1 ) and Lp(a) (≥50 mg dL-1 ) (OR 3.46, 95% CI 1.40-8.58, P = 0.007) or OxPL-apoB (≥2.02 nmol L-1 , median) (OR 5.48, 95% CI 2.45-12.27, P < 0.0001) had an elevated risk of CAVS. CONCLUSION: Autotaxin is a novel and independent predictor of CAVS in patients with CAD.

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.

Role of oxidized low density lipoprotein in atherogenesis.

Evidence to support an important role of oxidative modification in mediating the atherogenicity of LDL continues to grow. New hypotheses suggest mechanisms by which Ox-LDL or products of Ox-LDL can affect many components of the atherogenic process, including vasomotor properties and thrombosis, as well as lesion initiation and progression itself. These ideas suggest new approaches, that in combination with lowering of plasma cholesterol, could lead to the prevention of atherosclerosis and its complications.

A novel method for generating region-specific monoclonal antibodies to modified proteins. Application to the identification of human glucosylated low density lipoproteins.

Modifications of plasma lipoprotein structure and function resulting from in vivo post-translational nonenzymatic glycosylation may play a role in the premature atherosclerosis of patients with diabetes mellitus. This report describes the generation and characterization of six unique murine monoclonal antibodies that bind glucosylated human plasma lipoproteins, but do not react with normal plasma lipoproteins. This was accomplished by immunizing mice with homologous glucosylated low density lipoprotein. In competitive inhibition radioimmunoassays, the dominant epitope recognized by these antibodies on glucosylated low density lipoprotein was identified as glucitollysine, the reduced hexose alcohol form of glucose conjugated to the epsilon amino group of lysine. Each of these antibodies was capable of identifying glucitollysine epitopes on all reduced glucosylated proteins studied, including high density lipoprotein, albumin, hemoglobin, and transferrin. These antibodies were also capable of identifying and quantitating glucitollysine residues on the total plasma proteins and isolated lipoproteins of normal and diabetic individuals after reduction of the proteins with NaBH4. Preliminary data suggest that diabetic total plasma proteins and isolated lipoproteins contain at least threefold more immunochemically detectable glucitollysine residues than nondiabetic plasma proteins and lipoproteins. The technique described in this report should allow production of region-specific antibodies to any immunogenic modification of a protein.

Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload.

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.

Receptor-mediated catabolism of low density lipoprotein in man. Quantitation using glucosylated low density lipoprotein.

Low density lipoprotein (LDL) catabolism occurs by LDL receptor-dependent and LDL receptor-independent pathways. We have shown previously that nonenzymatic glucosylation of LDL in the presence of cyanoborohydride irreversibly blocks the lysine residues of LDL. Glucosylated LDL (GLC-LDL) was not degraded by the LDL receptor of fibroblasts, and its degradation by macrophages was similar to that of native LDL. This suggested that GLC-LDL should be a good tracer of LDL receptor-independent catabolism, and if combined with a tracer of total LDL catabolism, should enable one to calculate the extent of LDL receptor-dependent catabolism. To determine the contribution of each pathway in man, we prepared (125)I-GLC-LDL and (131)I-control LDL and simultaneously determined the fractional catabolic rate (FCR) of each tracer in four subjects. In preliminary experiments, we showed that the conditions for glucosylation did not affect LDL turnover. In the four subjects, the FCR for total LDL catabolism ranged from 0.345 to 0.724 d(-1) with a mean of 0.57+/-0.16 d(-1). The FCR of GLC-LDL varied from 0.071 to 0.141 d(-1) with a mean of 0.11+/-0.03 d(-1). The latter is similar to the FCR reported for native LDL in subjects with homozygous familial hypercholesterolemia, supporting the interpretation that GLC-LDL traces only the receptor-independent pathway. Despite the wide range of total LDL catabolism in these subjects. LDL receptor-independent catabolism accounted for only 19.5-20.6% of total catabolism. In turn, LDL receptor-dependent catabolism accounted for 80% of total clearance in each person. Furthermore, while the decay curve of LDL showed the usual biphasic pattern, the decay curve of GLC-LDL was monoexponential in each subject even when followed for as long as 48 d. This suggests that LDL receptor activity is responsible for the biphasic nature of LDL decay. These studies emphasize the central role of LDL receptor activity in normal LDL metabolism in man.

Structure, immunology, and cell reactivity of low density lipoprotein from umbilical vein of a newborn type II homozygote.

In this report we compare the cord blood lipoproteins of a newborn boy homozygote who has low density lipoprotein (LDL) receptor-defective familial hypercholesterolemia (FH) with the lipoproteins from cord blood of normal newborns. Plasma LDL-cholesterol and apoprotein (Apo)B were 612 and 233 mg/dl (vs. 31+/-16 and 24+/-12 mg/dl, respectively, for normals, n = 21). LDL-cholesterol/ApoB ratio was 2.6 vs. 1.4+/-0.5. Levels of ApoA-I, ApoA-II, and HDL-cholesterol were similar to normal cord plasma. Thus, the lipoprotein abnormality is apparent at birth and is definitely present in LDL. Abnormalities in other lipoprotein, lipid, and in plasma apoprotein levels were not detected. On zonal ultracentrifugation, FH LDL was comprised of two populations (LDL(a) and LDL(b)), both faster floating than normal cord LDL (LDL(c)). This difference was due to the larger diameters of the particles on electron microscopy (LDL(a) = 276A+/-32 and LDL(b) = 260A+/-38 vs. LDL(c) = 237A+/-26, n = 200 each, mean+/-1 SD), and their higher contents of lipids relative to protein (86 and 82% vs. 74%, LDL(a), LDL(b), and LDL(c), respectively). More than 94% of the protein in both the FH and the normal preparations consisted of ApoB. FH LDL were more effective than control LDL in competing with (125)I-LDL (adult) for limiting amounts of anti-LDL antibodies in radioimmunoassay. FH LDL also competed more effectively for binding to LDL receptors on cultured fibroblasts at 4 degrees C, and FH LDL also delivered more cholesterol into the cells. Cells grown in lipoprotein-deficient serum contained 44+/-2 mug cholesterol/mg cell protein, incubation of cells for 18 h at 37 degrees C in 5 mug/ml FH LDL (protein) or in normal LDL raised cellular cholesterol levels to 75+/-2 and 60+/-2 mug/mg, respectively.LDL isolated from the FH patient's plasma at 6 mo of age and from his brother's plasma (a 5-yr-old boy FH homozygote) were similar to LDL isolated from normolipemic subjects in flotation properties, chemical composition, and immunochemical and cell reactivity. The fact that differences between normal cord LDL and FH cord LDL were present at birth, but that the differences between control and FH LDL were no longer present postnatally suggests that the altered immunologic and cell interactive properties of FH cord LDL were probably related to its unusually high contents of core lipids.

Characterization of an abnormal species of apolipoprotein B, apolipoprotein B-37, associated with familial hypobetalipoproteinemia.

Steinberg and colleagues have previously described a unique kindred with normotriglyceridemic hypobetalipoproteinemia (1979. J. Clin. Invest. 64:292-301). In a reexamination of this kindred, we found an abnormal apolipoprotein (apo) B species, apo B-37 (203,000 mol wt), in the plasma lipoproteins of multiple members of the kindred. In affected individuals apo B-37 was found in very low density lipoproteins, along with the normal apo B species, apo B-100 and apo B-48. High density lipoproteins (HDL) also contained apo B-37, but no other apo B species. The first 13 amino-terminal amino acids of apo B-37 were identical to those of normal apo B-100. We utilized a panel of 18 different apo B-specific monoclonal antibodies and polyclonal antisera specific for apo B-37 and the thrombin cleavage products of apo B-100 to map apo B-37 in relation to apo B-100, apo B-48, and the thrombin cleavage products of apo B-100. The results of those immunochemical studies indicated that apo B-37 contains only amino-terminal domains of apo B-100. In affected individuals, the majority of apo B-37 in plasma was contained in the HDL density fraction. Within that fraction apo B-37 was found on discrete lipoprotein particles, termed Lp-B37, that had properties distinct from normal HDL particles containing apo A-I. This report documents for the first time the existence of an abnormal apo B species in humans. Further study of apo B-37 and lipoprotein particles containing apo B-37 should lead to an improved understanding of apo B structure and function.

Diversity in expression of heterozygous familial hypercholesterolemia. Characterization of a unique kindred.

Clinical and biochemical characteristics of familial hypercholesterolemia (FH) heterozygotes possessing an abnormally high molecular weight low density lipoprotein receptor (HMWR) are reported. The disorder is transmitted as an autosomal dominant trait and is not distinguishable from classic heterozygous FH on clinical grounds. The average plasma low density lipoprotein (LDL) level is 360 mg/dl and tendon xanthomata and early coronary disease are present. LDL receptor activity is higher than expected. In skin fibroblast cultures two types of functional LDL receptors are present, one with a normal apparent native molecular weight of 140,000, and the other of 176,000. When immobilized on nitrocellulose paper both receptors bind LDL. Maximum 125I-LDL binding capacity of fibroblast monolayers is reduced only 20%, compared with 50% in typical heterozygous FH. Affinity for 125I-LDL is increased and a 38% reduction in the Michaelis constant for LDL is observed. When autologous 125I-LDL was injected intravenously, the fractional catabolic rate of LDL was 205% and the LDL apoprotein B production rate was 328% of that found in a typical heterozygous FH subject. Thus, both in vitro and in vivo testing indicated only a modest deficiency of LDL receptor activity. Kindred members possessing the HMWR had an associated abnormality of cholesterol biosynthesis. Cholesterol balance studies in three individuals with the HMWR trait demonstrated elevated cholesterol biosynthesis of two to three times the mean of normal subjects. These findings suggest that increased LDL production and increased cholesterol production may assume a significant role in the pathologic manifestations of heterozygous FH. Functional abnormalities in LDL receptor activity as measured in fibroblast culture may be relatively small.

Lipolysis produces changes in the immunoreactivity and cell reactivity of very low density lipoproteins.

Smaller very low density lipoprotein (VLDL) remnants interact more readily with tissues than do larger "intact" VLDL. This may be related to changes in the availability of VLDL apoproteins on the surface of the lipoproteins. To test this hypothesis VLDL were incubated at 37 degrees C with bovine milk lipase (LPL), and the abilities of LPL-treated VLDL preparations to compete with (125)I-low density lipoproteins (LDL) for interaction with cultured normal human fibroblasts were measured. At the same time, the immunologic activities of these preparations were also tested by double antibody radioimmunoassay. Triglyceride (TG) contents of VLDL fell by 30-90% during incubation with LPL and, on zonal ultracentrifugation, VLDL of faster Svedberg unit of flotation (S(f1.063)) rates (>150) were gradually converted to smaller VLDL with lower S(f) rates (21-60). LPL-treated VLDL competed two to five times more effectively with (125)I-LDL for binding to cellular receptors than did control VLDL. Control VLDL incubated with heat-inactivated LPL at 37 degrees C, or with active LPL at 4 degrees C had unaltered cell reactivities and TG contents compared with VLDL incubated without any enzyme. The direct uptake and degradation of LPL-treated VLDL was also assessed by using VLDL (125)I-labeled in apoprotein (Apo)B. LPL-treated VLDL-(125)I-ApoB were taken up and degraded by fibroblast at greater rates than were control VLDL-(125)I-ApoB. Thus, hydrolysis of VLDL lipids was accompanied by an increased ability of VLDL to interact with fibroblasts. The immunoreactivity of ApoB in the same VLDL preparations, expressed as the "apparent ApoB contents" of LPL-treated VLDL, increased by 10-50% (P < 0.02) in those assays that contained anti-LDL antisera, but the ApoB of control VLDL remained constant. However, assays that contained antisera directed against ApoB isolated from VLDL did not distinguish between LPL-treated and control VLDL. Thus, VLDL lipid hydrolysis was accompanied by changes in the immunoreactivity of VLDL-ApoB, which probably reflect changes in the disposition of ApoB on the surface of VLDL. The altered disposition of ApoB on VLDL "remnants" may be related to their enhanced interaction with cells.

Comparison of glucosylated low density lipoprotein with methylated or cyclohexanedione-treated low density lipoprotein in the measurement of receptor-independent low density lipoprotein catabolism.

We previously showed that glucosylation of lysine residues of low density lipoproteins (LDL) blocks high-affinity degradation by cultured human fibroblasts, and markedly slows LDL turnover in guinea pigs. The present studies were done to evaluate glucosylated (GLC) LDL as a tracer of receptor-independent LDL catabolism, and to compare it with two other modified LDL, methylated (MET) LDL, and cyclohexanedione (CHD)-treated LDL, which have been used previously for this purpose. Glucosylation of LDL did not affect receptor-independent degradation in vivo, as the turnover of GLC-LDL and native LDL were similar in the LDL receptor-deficient, Watanabe heritable hyperlipidemic rabbit. Each modified radiolabeled LDL preparation was injected into eight guinea pigs, and fractional catabolic rates (FCR) determined. The FCR of GLC-LDL (0.024 +/- 0.005 h-1; SD) was similar to that of MET-LDL (0.023 +/- 0.006 h-1), and approximately 22% of that of native LDL (0.105 +/- 0.02 h-1). The FCR of CHD-LDL was greater than that of the other modified LDL, and it varied depending on how soon after preparation the CHD-LDL was injected: when used within 2 h of preparation, the mean FCR was 0.044 +/- 0.007 h-1 (n = 4); when used after overnight dialysis at 4 degrees C, the mean FCR was 0.082 +/- 0.03 h-1 (n = 4). This suggests that CHD-LDL overestimates the amount of LDL degraded by receptor-independent pathways, perhaps because the CHD modification is spontaneously reversible. The present studies indicate that GLC-LDL is a useful tracer of receptor-independent LDL catabolism in animals.

Familial hypercholesterolemia. Evidence for a newly recognized mutation determining increased fibroblast receptor affinity but decreased capacity for low density lipoprotein in two siblings.

Cultured skin fibroblasts were obtained from two siblings with classic clinical features of homozygous familial hypercholesterolemia. Plasma cholesterol values were 970 and 802 mg/100 ml in the siblings, 332 mg/100 ml in the mother, and 426 mg/100 ml in the father. Fibroblast receptor-specific capacity for binding and degradation of (125)I-low density lipoprotein (LDL) at 37 degrees C was 11% of normal, consistent with the diagnosis of "homozygous LDL receptor-defective" hypercholesterolemia, a disorder in which LDL binding activity is low but detectable. The residual LDL receptor activity was clearly qualitatively abnormal. The Michaelis constant (K(m)) for (125)I-LDL was reduced to 20-40% of normal, indicating a substantially increased affinity for LDL. Increased affinity and reduced capacity for (125)I-LDL are also found when normal fibroblasts are assayed at 4 degrees C. As the temperature is raised to 37 degrees C surface LDL binding affinity decreases while capacity increases. At 4 degrees C the fibroblasts of our subjects had an affinity for LDL indistinguishable from normal cells assayed at that temperature and a binding capacity 23% of normal. However, only small changes in affinity and capacity occurred upon increasing the temperature to 37 degrees C. When (125)I-apoprotein E-phospholipid vesicles were bound at 37 degrees C the receptor deficiency appeared only half as severe as when (125)I-LDL was used as ligand.A family study suggests that the siblings are genetic compounds rather than homozygotes, having inherited a mutant maternal gene causing absent or silent LDL receptors and a mutant paternal gene resulting in qualitatively altered LDL receptors. It is not clear whether these defects are present at the same or different genetic loci. The altered receptors are characterized by increased affinity and moderately reduced capacity for LDL at 37 degrees C and are accompanied by hypercholesterolemia at least as severe as that associated with familial hypercholesterolemia with absent or nonfunctional LDL receptors.

Probucol inhibits oxidative modification of low density lipoprotein.

Previous studies have established that low density lipoprotein (LDL) incubated with endothelial cells (EC) undergoes extensive oxidative modification in structure and that the modified LDL is specifically recognized by the acetyl LDL receptor of the macrophage. Thus, in principle, EC-modified LDL could contribute to foam cell formation during atherogenesis. Oxidatively modified LDL is also potentially toxic to EC. The present studies show that addition of probucol during the incubation of LDL with EC prevents the increase in the electrophoretic mobility, the increase in peroxides, and the increase in subsequent susceptibility to macrophage degradation. It has also been shown that oxidation of LDL catalyzed by cupric ion induces many of the same changes occurring during EC modification. Addition of probucol (5 microM) also prevented this copper-catalyzed modification of LDL. Most importantly, samples of LDL isolated from plasma of hypercholesterolemic patients under treatment with conventional dosages of probucol were shown to be highly resistant to oxidative modification either by incubation with endothelial cells or by cupric ion in the absence of cells. The findings suggest the hypothetical but intriguing possibility that probucol, in addition to its recognized effects on plasma LDL levels, may inhibit atherogenesis by limiting oxidative LDL modification and thus foam cell formation and/or EC injury. Other compounds with antioxidant properties might behave similarly.

Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

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.

Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts.

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.

Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions.

To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more and larger lesions (758,651+/-87,449 and 451,255+/-37,448 micron2 per section, respectively; mean+/-SD) than aortas from normocholesterolemic mothers (61,862+/-9,555 micron2; P < 0.00005). Serial sections of the arch, thoracic, and abdominal aortas were immunostained for recognized markers of atherosclerosis: macrophages, apo B, and two different oxidation-specific epitopes (malondialdehyde- and 4-hydroxynonenal-lysine). Of the atherogenic sites that showed positive immunostaining for at least one of these markers, 58.6% were established lesions containing both macrophage/foam cells and oxidized LDL (OxLDL). 17.3% of all sites contained only native LDL, and 13.3% contained only OxLDL without monocyte/ macrophages. In contrast, only 4.3% of sites contained isolated monocytes in the absence of native or oxidized LDL. In addition, 6.3% of sites contained LDL and macrophages but few oxidation-specific epitopes. These results demonstrate that LDL oxidation and formation of fatty streaks occurs already during fetal development, and that both phenomena are greatly enhanced by maternal hypercholesterolemia. The fact that in very early lesions LDL and OxLDL are frequently found in the absence of monocyte/macrophages, whereas the opposite is rare, suggests that intimal LDL accumulation and oxidation contributes to monocyte recruitment in vivo.

Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.

Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynonenal-lysine adducts in glomeruli of PHN rats by immunofluorescence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by approximately 85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria. These findings are consistent with the premise that ROS-induced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with proteinuria and the ability of probucol to inhibit proteinuria support a causal role for LPO in the the alteration of the glomerular permselectivity which results in proteinuria.

Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma.

Many reactive products may be formed when LDL undergoes lipid peroxidation, which in turn can react with lipids, apoproteins, and proteins, generating immunogenic neoepitopes. Autoantibodies recognizing model epitopes of oxidized low density lipoprotein, such as malondialdehydelysine, occur in plasma and in atherosclerotic lesions of humans and animals. Because apo E-deficient mice develop particularly high titers of such autoantibodies, we used their spleens to clone 13 monoclonal antibodies to various epitopes of oxidized LDL ("E0 antibodies"). Binding and competitive RIAs demonstrated significant differences in fine specificity even between E0 antibodies initially selected for binding to the same screening antigen. For example, some E0 antibodies selected for binding to malondialdehyde-LDL also recognized copper oxidized LDL, acrolein-LDL, or LDL modified by arachidonic or linoleic acid oxidation products. Circulating IgG and IgM autoantibodies binding to copper-oxidized LDL, 4-hydroxynonenal-LDL, acrolein-LDL, and LDL modified with arachidonic or linoleic acid oxidation products were found in apo E-deficient mice, suggesting that the respective antigens are formed in vivo. Epitopes recognized by some of the E0 monoclonal antibodies were also found on human circulating LDL. Each of the E0 monoclonal antibodies immunostained rabbit and human atherosclerotic lesions, and some of them yielded distinct staining patterns in advanced lesions. Together, this suggests that the natural monoclonal antibodies recognize different epitopes of complex structures formed during oxidation of lipoproteins, or epitopes formed independently at different lesion sites. Our data demonstrate that a profound immunological response to a large number of different epitopes of oxidized lipoproteins occurs in vivo. The availability of "natural" monoclonal autoantibodies should facilitate the identification of specific epitopes inducing this response.

Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity.

The immune response to oxidized LDL (OxLDL) may play an important role in atherogenesis. Working with apoE-deficient mice, we isolated a panel of OxLDL-specific B-cell lines that secrete IgM Abs that specifically bind to oxidized phospholipids such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC). These Abs block uptake of OxLDL by macrophages, recognize similar oxidation-specific epitopes on apoptotic cells, and are deposited in atherosclerotic lesions. The Abs were found to be structurally and functionally identical to classic "natural" T15 anti-PC Abs that are of B-1 cell origin and are reported to provide optimal protection from virulent pneumococcal infection. These findings suggest that there has been natural selection for B-1 cells secreting oxidation-specific/T15 antibodies, both for their role in natural immune defense and for housekeeping roles against oxidation-dependent neodeterminants in health and disease.

Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects.

We report the results of feeding oleate- or linoleate-enriched diets for 8 wk to mildly hypercholesterolemic subjects and the resulting alterations in composition and functional properties of their plasma LDL and HDL. LDL isolated from subjects on oleate-enriched diets was less susceptible to copper-mediated oxidation, as measured by conjugated diene and lipid peroxide formation, and less susceptible to LDL-protein modification, as evidenced by reduced LDL macrophage degradation after copper- or endothelial cell-induced oxidation. For all subjects, the percentage of 18:2 in LDL correlated strongly with the extent of conjugated diene formation (r = 0.89, P < 0.01) and macrophage degradation (r = 0.71, P < 0.01). Oxidation of LDL led to initial rapid depletion of unsaturated fatty acids in phospholipids followed by extensive loss of unsaturated fatty acids in cholesteryl esters and triglycerides. Changes in HDL fatty acid composition also occurred. However, HDL from both dietary groups retained its ability to inhibit oxidative modification of LDL. This study demonstrates that alterations in dietary fatty acid composition can effectively alter the fatty acid distribution of LDL and HDL in hypercholesterolemic subjects and that susceptibility to LDL oxidation is altered by these changes. Substitution of monounsaturated (rather than polyunsaturated) fatty acids for saturated fatty acids in the diet might be preferable for the prevention of atherosclerosis.