Development of Capture Assays for Different Modifications of Human Low-Density Lipoprotein

Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), N(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r = 0.706, P < 0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.     

Clinical significance of the humoral immune response to modified LDL

Human low density lipoprotein (LDL) undergoes oxidation and glycation in vivo. By themselves, oxidized LDL (oxLDL) and AGE-LDL have proinflammatory properties and are considered atherogenic. But the atherogenicity of these lipoproteins are significantly increased as a consequence of the formation of immune complexes (IC) involving specific autoantibodies. OxLDL and AGE antibodies have been shown to be predominantly of the IgG1 and IgG3 isotypes. OxLDL antibodies are able to activate the complement system by the classical pathway and to induce FcR-mediated phagocytosis. In vitro and ex vivo studies performed with modified LDL-IC have proven their pro-inflammatory and atherogenic properties. Clinical studies have demonstrated that the levels of circulating modified LDL-IC correlate with parameters indicative of cardiovascular and renal disease in diabetic patients and other patient populations. The possibility that spontaneously formed or induced modified LDL antibodies (particularly IgM oxLDL antibodies) may have a protective effect has been suggested, but the data is unclear and needs to be further investigated.     

Anti-oxidized low-density lipoprotein antibodies in myeloperoxidase-positive vasculitis patients preferentially recognize hypochlorite-modified low density lipoproteins

Many patients surviving vasculitis are prone to accelerated atherosclerosis and often have enhanced levels of antibodies to oxidized low-density lipoprotein (oxLDL). To measure anti-oxLDL antibodies, oxidation of LDL is achieved with copper (Cu) or malondialdehyde (MDA). Because, in vivo, LDL may be oxidized with myeloperoxidase (MPO) or its product hypochlorite, we measured anti-hypochlorite LDL antibodies in patients with vasculitis, haemodialysis patients and healthy controls. A newly developed enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to oxLDL as modified by hypochlorite. Results are compared with data obtained by standard LDL oxidation using MDA-LDL or Cu-LDL as substrate. Results were compared between anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients (n = 93), haemodialysis (HD) patients (n = 59) and healthy controls (HC; n = 43). Furthermore, patients with MPO-ANCA-associated vasculitis (n = 47) were compared with patients with proteinase 3 (PR3)-ANCA associated vasculitis (n = 46). Optimal cut-off points were determined by receiver operator characteristic (ROC) curve analysis. Anti-oxLDL antibodies are enhanced in AAV patients (MDA-LDL and hypochlorite-LDL) and in HD patients (hypochlorite-LDL), when compared to HC. Furthermore, patients with MPO-ANCA-associated vasculitis had higher levels of antibodies to hypochlorite-LDL than patients with PR3-ANCA-associated vasculitis. Our newly developed assay, in which hypochlorite-LDL is used as substrate, seems a more sensitive assay than traditional assays to measure oxLDL antibodies. Furthermore, our results suggest that enhanced MPO-mediated LDL oxidation occurs in patients with MPO-ANCA.     

Immunochemical characterization of purified human oxidized low-density lipoprotein antibodies

The goal of this study was to characterize the isotypes and reactivity of human autoantibodies to copper oxidized LDL (oxLDL). Forty-six purified oxLDL antibodies contained immunoglobulins of the three major isotypes, with a predominance of IgG, subclasses 1 and 3. These IgG isotypes are known to interact with FcRgammaI and to activate the complement system and thus are potentially able to activate macrophages and cause foam cell formation. The same purified antibodies were tested for cross-reactivity with malondialdehyde (MDA)-, glycated (Glyc)-, and native (n)LDL and cardiolipin. Absorption with oxLDL resulted in a decrease of reactivity of 77.2 +/- 4.7%. Absorption with MDA-LDL resulted in a wider range of reduction of reactivity values, ranging from 50 to 87%, possibly reflecting differences in the degree of MDA modification. Absorption with Glyc- and nLDL caused a minor decrease in the reactivity of antibodies to oxLDL (5.9 +/- 7.1 and 6.8 +/- 6. 4%, respectively), comparable to the reduction of reactivity (2.1 +/- 4.0%) measured after absorption with transferrin, an irrelevant protein used as a negative control. These results suggest that oxLDL antibodies recognize primarily MDA epitopes. To determine whether purified oxLDL antibodies also recognize other epitopes known to be generated during copper oxidation of LDL, such as 4-hydroxynonenal (HNE)- and N(epsilon)(carboxymethyl)-lysine (CML), two additional sets of experiments were carried out. First, we monitored the formation of CML-, MDA-lysine, and HNE-lysine at different times during copper oxidation of two LDL pools. Both pools showed simultaneous increases in protein modification, as indicated by increasing fluorescence emission at 430 nm, and in immunoreactivity with oxLDL antibodies, coinciding closely with MDA modification of lysine groups. Second, we assessed whether the reactivity of oxLDL antibodies could be blocked by absorption with CML- or HNE-LDL. HNE-LDL did not react with isolated oxLDL antibodies. Highly modified CML-LDL (>90% of lysine residues modified) reduced the reactivity of oxLDL antibodies, but only by 25.5%. Finally, we investigated the possible cross-reactivity of oxLDL antibodies with cardiolipin. Seventeen purified oxLDL antibodies were used in this study, which showed that absorption with oxLDL or nLDL did not affect their reactivity with immobilized cardiolipin.     

Proatherogenic and proinflammatory properties of immune complexes prepared with purified human oxLDL antibodies and human oxLDL

Immune complexes (IC) prepared with human low density lipoprotein (LDL) and rabbit LDL antibodies induce foam cell transformation of human macrophages and activate the release of proinflammatory mediators by human macrophages and THP-1 cells. Because the affinity of human oxidized LDL (oxLDL) antibodies is lower than that of rabbit antibodies, IC formed with human antibodies could have limited pathogenic potential. Immune complexes prepared with human oxidized LDL (oxLDL) and purified human oxLDL antibodies (predominantly of the IgG1 and IgG3 isotypes) were presented to THP-1 cells using two protocols previously described in studies of the properties of LDL-IC prepared with rabbit antibodies. OxLDL/human oxLDL antibody IC immobilized by adsorption to red blood cells (RBC) induced the release of significantly higher levels of TNF from THP-1 cells (872-313 pg/ml) than oxLDL adsorbed to RBC (461-75.6 pg/ml) and caused a higher degree of cholesterol ester accumulation in the same cells (5.4-0.77 in cells incubated with IC-coated RBC vs 1.99-1.16 in oxLDL-coated RBC). Insoluble IC prepared with oxLDL/human oxLDL antibody were even more effective in promoting intracellular accumulation of cholesterol in THP-1 cells (total cholesterol = 53.8-13.5 and cholesterol esters = 24.0-7.2 mg/l in THP-1 cells incubated with insoluble IC (200 micrograms) vs total cholesterol = 32.4-8.2 and cholesterol esters = 7.7 +/- 2.8 micrograms/l in THP-1 cells incubated with an identical concentration of oxLDL) and also induced the release of TNF. Thus we have demonstrated that IC prepared with human oxLDL and human oxLDL antibodies have the same atherogenic and proinflammatory properties as IC prepared with human LDL and rabbit LDL antibodies. This strongly supports the concept that modified LDL-IC present in circulation and/or tissues play an important pathogenic role in arteriosclerosis.     

Immune complex-like activity associated with abnormal serum lipoproteins in systemic erythematosus.

Sera from four patients with systemic lupus erythematosus (SLE) were shown to contain abnormal lipoproteins which behaved as immune complexes (IC). One IC lipoprotein (IC VLDL) had the density in ultracentrifugation of very low density lipoprotein, but a markedly altered electrophoretic mobility. A second IC lipoprotein (IC LDL) had the electrophoretic mobility of very low density lipoprotein but was slightly denser than low density lipoprotein on ultracentrifugation. Both the IC VLDL and IC LDL contained IgG and behaved as IC in the Clq deviation test, platelet aggregation and rheumatoid factor inhibition assays, but not in the conglutinin and Clq binding assays and the Clq solid phase assay. These differences could be due to the low densities of the IC VLDL and IC LDL. The abnormal lipoprotein IC disappeared with clinical remission in two patients and were not present in the sera of other patients with inactive or mild SLE, Type IV hyperlipidaemia, or during prednisone therapy or plasma exchange for other conditions. These IC appeared to be markers of severe and active SLE.     

Phosphorylcholine-dependent cross-reactivity between dental plaque bacteria and oxidized low-density lipoproteins

Antibodies reactive with phosphorylcholine (PC) are ubiquitous in human sera, but the antigens stimulating their production and their function are not clear. Previous studies have shown that a significant proportion of dental plaque bacteria contain PC as determined by reactivity with PC-specific mouse myeloma proteins and monoclonal antibodies. Additionally, serum antibody concentrations of immunoglobulin (IgG) G anti-PC are higher in sera of individuals who have experienced periodontal attachment loss than those who are periodontally healthy. These data implicate the oral microflora as a source of antigen-stimulating anti-PC responses. Recent data also indicate that antibodies with specificity for PC are elevated in ApoE-deficient mice, a model for studies of athersclerosis, and that such antibodies bound oxidized low-density lipoproteins (LDL) (oxLDL) in atherosclerotic plaques. These data prompted the hypothesis that human anti-PC could bind to both oral bacteria and human oxLDL, and that these antigens are cross-reactive. We therefore examined the ability of human anti-PC to bind to PC-bearing strains of oral bacteria using enzyme-linked immunosorbent inhibition assays and by assessment of direct binding of affinity-purified human anti-PC to PC-bearing Actinobacillus actinomycetemcomitans. Our results indicated that PC-bearing strains of Streptococcus oralis, Streptococcus sanguis, Haemophilus aphrophilus, Actinomyces naeslundii, Fusobacterium nucleatum, and A. actinomycetemcomitans, as well as a strain of Streptococcus pneumoniae, absorbed up to 80% of anti-PC IgG antibody from human sera. Furthermore, purified anti-PC bound to a PC-bearing strain of A. actinomycetemcomitans but only poorly to a PC-negative strain. OxLDL also absorbed anti-PC from human sera, and oxLDL but not LDL reacted with up to 80% of the anti-PC in human sera. Furthermore, purified anti-PC bound directly to oxLDL but not to LDL. The data indicate that PC-containing antigens on a variety of common oral bacteria are cross-reactive with neoantigens expressed in oxLDL. We propose that PC-bearing dental plaque microorganisms may induce an antibody response to PC that could influence the inflammatory response associated with atherosclerosis.     

Apolipoprotein B100 is a suppressor of Staphylococcus aureus‐induced innate immune responses in humans and mice

Plasma lipoproteins such as LDL (low‐density lipoprotein) are important therapeutic targets as they play a crucial role in macrophage biology and metabolic disorders. The impact of lipoprotein profiles on host defense pathways against Gram‐positive bacteria is poorly understood. In this report, we discovered that human serum lipoproteins bind to lipoteichoic acid (LTA) from Staphylococcus aureus and thereby alter the immune response to these bacteria. Size‐exclusion chromatography and solid‐phase‐binding analysis of serum revealed the direct interaction of LTA with apolipoproteins (Apo) B100, ApoA1, and ApoA2. Only ApoB100 and the corresponding LDL exerted biological effects as this binding significantly inhibited LTA‐induced cytokine releases from human and murine immune cells. Serum from hypercholesterolemic mice or humans significantly diminished cytokine induction in response to S. aureus or its LTA. Sera taken from the patients with familial hypercholesterolemia before and after ApoB100‐directed immuno‐apheresis confirmed that ApoB100 inhibited LTA‐induced inflammation in humans. In addition, mice in which LDL secretion was pharmacologically inhibited, displayed significantly increased serum cytokine levels upon infection with S. aureus in vivo. The present study identifies ApoB100 as an important suppressor of innate immune activation in response to S. aureus and its LTA.     

Distribution of IgM and IgG antibodies to oxidized LDL in immune complexes isolated from patients with type 1 diabetes and its relationship with nephropathy

Modified lipoproteins are immunogenic and play a key pathogenic role in vascular disease. Antibodies to oxidized LDL (oxLDL) are mostly of the pro-inflammatory IgG₁ and IgG₃ isotypes. We measured IgG and IgM oxLDL antibodies in immune complexes (IC) isolated from 36 patients with type 1 diabetes using a nested case control design. IgG antibodies predominated over IgM antibodies by an 8:1 ratio. IgG antibody concentrations were higher in the nephropathy cases compared to controls (p = 0.09), but no significant difference was observed because of two patients included in the study who had end-stage renal disease (creatinine > 5 mg/dL and glomerular filtration rate (GFR) less than 17 mL/min). After eliminating these patients from the analysis, significant positive associations of IgG antibody concentration with serum creatinine and albumin excretion rate were observed. Similarly, a negative correlation with estimated glomerular filtration rate was observed in this subsample of 34 patients. Differences in IgM antibody concentrations by nephropathy classification were not supported by the data. In conclusion, the predominance of pro-inflammatory IgG oxLDL antibodies is associated with existence of diabetic nephropathy, and a protective role of IgM antibodies could not be demonstrated.     

Imaging of aortic atherosclerotic lesions by (125)I-LDL, (125)I-oxidized-LDL, (125)I-HDL and (125)I-BSA.

OBJECTIVE: The aim of the present study was to compare the accumulation of (125)I-labeled low-density lipoproteins (LDL), oxidized LDL (oxLDL), HDL and BSA in advanced atherosclerotic lesions of apoE-deficient mice. METHODS: (125)I-lipoproteins or (125)I-BSA were injected into the tail vein of apoE-deficient mice. Blood clearance of (125)I-lipoproteins and (125)I-BSA and their accumulation in atherosclerotic lesions were assayed. RESULTS: Blood clearance of (125)I-LDL and (125)I-HDL was moderate, and approximately 30% of the injected lipoproteins were present in plasma 24 h following injection. oxLDL was removed much faster from plasma, and less than 10% of (125)I-oxLDL was present in the circulation 30 min after (125)I-oxLDL injection. The clearance of (125)I-BSA from the circulation was slower than the lipoprotein clearance. The highest accumulation of LDL, oxLDL, HDL and BSA was detected in atherosclerotic lesions in the aortic arch and abdominal aorta, while lower accumulation was detected in the less atherosclerotic descending thoracic aorta. CONCLUSION: Our findings demonstrate that both (125)I-HDL and (125)I-BSA as well as (125)I-LDL are accumulated in atherosclerotic plaques and that they can be used for the detection of atherosclerotic lesions.     

Analysis of the long-term efficacy and selectivity of immunoadsorption columns for low density lipoprotein apheresis

Immunoadsorption low density lipoprotein (LDL) apheresis is performed with reusable columns containing anti-apolipoprotein B(ApoB) antibodies. We analyzed their long-term efficacy and selectivity. Performance over 60 treatment sessions of six pairs of immunoadsorption LDL apheresis columns was evaluated by analysis of variance using the removal of total cholesterol and ApoB to assess efficacy and the ratio of total cholesterol/high density cholesterol removed to assess selectivity. The removal of cholesterol did not vary significantly with treatment number. The mass of ApoB removed increased significantly (p = 0.002), and the mass of ApoB removed per volume unit of processed plasma showed a trend (p = 0.065) toward an increase with treatment number. Both parameters correlated with the serum ApoB concentration before treatment, which also increased significantly (p = 0.0007) with treatment number. No significant variation of selectivity was found. The efficacy of the LDL apheresis immunoadsorption columns did not decrease after 60 treatment sessions. The columns' selectivity also remained unchanged.     

The binding of human low-density lipoproteins to the surface of schistosomula of Schistosoma mansoni is inhibited by polyanions and reduces the binding of anti-schistosomal antibodies.

Host molecules such as serum lipoproteins, blood group glycolipids, and histocompatibility antigens may bind to schistosomes and thereby prevent immune recognition of the parasite. This study examines the kinetics of lipoprotein binding, the ability of polyanions to inhibit lipoprotein binding, the binding of anti-schistosomal antibodies to worms that have previously bound low-density lipoprotein (LDL), and the distribution of lipoproteins bound to the parasites. Lipoproteins in human serum (HS) and purified LDL, very low-density lipoprotein (VLDL), and apolipoprotein B (apo B) in defined media were demonstrated on the surface of schistosomula of Schistosoma mansoni by fluorescence and immunoelectron microscopy using a polyclonal goat anti-human apolipoprotein B antibody (anti-apo B). By fluorophotometric microscopy, lipoprotein binding began within 15 minutes and was largely completed within 3 hours of exposure. Lipoprotein binding saturated at 10% HS or 20 micrograms protein/300 microliters of purified LDL. Suramin inhibited LDL binding by 59% in a dose-dependent fashion. In the absence of LDL in the medium, 2 mM suramin dissociated 41% of bound LDL from the worm surface within 15 minutes and 10 mg/ml heparin dissociated 36%. The binding of human anti-schistosomal antibodies to schistosomula was inhibited by bound LDL. By fluorescence microscopy, serum or purified lipoproteins were distributed over the entire surface of the parasite with focal areas of high intensity. Ultrastructurally, reaction product was seen on the outer leaflet of the outer tegumental membrane and in aggregates and surrounding vesicular structures varying in diameter from 13 to 83 nm. These studies demonstrate that lipoproteins bind to the surface of schistosomula. The binding of lipoproteins is partially inhibited by polyanions, reduces the binding of human anti-schistosomal antibodies, and may help the parasite escape the immune response.     

Human monoclonal Fab and human plasma antibodies to carbamyl‐epitopes cross‐react with malondialdehyde‐adducts

Oxidized low‐density lipoprotein (OxLDL) plays a crucial role in the development of atherosclerosis. Carbamylated LDL has been suggested to promote atherogenesis in patients with chronic kidney disease. Here we observed that plasma IgG and IgM antibodies to carbamylated epitopes were associated with IgG and IgM antibodies to oxidation‐specific epitopes (ρ = 0·65–0·86, P < 0·001) in healthy adults, suggesting a cross‐reaction between antibodies recognizing carbamyl‐epitopes and malondialdehyde (MDA)/malondialdehyde acetaldehyde (MAA) ‐adducts. We used a phage display technique to clone a human Fab antibody that bound to carbamylated LDL and other carbamylated proteins. Anti‐carbamyl‐Fab (Fab106) cross‐reacted with oxidation‐specific epitopes, especially with MDA‐LDL and MAA‐LDL. We showed that Fab106 bound to apoptotic Jurkat cells known to contain these oxidation‐specific epitopes, and the binding was competed with soluble carbamylated and MDA‐/MAA‐modified LDL and BSA. In addition, Fab106 was able to block the uptake of carbamyl‐LDL and MDA‐LDL by macrophages and stained mouse atherosclerotic lesions. The observed cross‐reaction between carbamylated and MDA‐/MAA‐modified LDL and its contribution to enhanced atherogenesis in uraemic patients require further investigation.     

Increased levels of low-density lipoprotein oxidation in patients with familial hypercholesterolemia and in end-stage renal disease patients on hemodialysis

Patients with familial hypercholesterolemia (FH) and patients with end-stage renal disease (ESRD) undergoing dialysis suffer from accelerated atherosclerosis. Oxidation of low-density lipoprotein (LDL) cholesterol is crucial in atherogenesis. In the present study, we determined the LDL oxidation level and oxidizability of isolated LDL of 11 male patients with FH, 15 male ESRD patients on hemodialysis, and 15 age-matched male normolipidemic healthy controls. FH patients were without lipid-lowering medication for at least 4 weeks and were reassessed after 2 years of cholesterol-lowering therapy (statins). LDL oxidation level was measured by ELISA using monoclonal antibody 4E6 to oxidized LDL (oxLDL) as the capture antibody and anti-human apoB antibody for detection; results were expressed as percentage oxLDL. In FH patients and in ESRD patients on hemodialysis, both groups having a higher percentage of cardiovascular disease, mean plasma LDL oxidation levels were significantly elevated compared with controls (4.9 +/- 1.3; 3.7 +/- 2.0; 1.7 +/- 0.6%, respectively). Within each group of subjects, LDL oxidation level was not associated with history of cardiovascular disease. Furthermore, in neither group was a significant correlation found between plasma concentration of LDL cholesterol and LDL oxidation level. After cholesterol-lowering therapy, LDL oxidation level in FH patients had not changed significantly and remained elevated compared with controls, despite a reduction of LDL cholesterol by 55% on average. Also, absolute plasma oxLDL concentrations, obtained by multiplying LDL oxidation level with plasma LDL cholesterol concentration, were significantly higher in FH patients before and after cholesterol-lowering therapy and in ESRD patients on hemodialysis than in controls (489 +/- 145; 189 +/- 122; 100 +/- 65; and 59 +/- 27 micro moles/L, respectively). No correlation was found between plasma oxLDL concentration and parameters of LDL oxidizability, LDL fatty acids, and LDL alpha-tocopherol content. We conclude that cholesterol-lowering therapy does not normalize elevated LDL oxidation levels in FH patients and elevated LDL oxidation level in FH and in ESRD might mirror atherosclerosis.     

Evidence for the presence in human plasma of lecithin: cholesterol acyltransferase activity (beta-LCAT) specifically esterifying free cholesterol of combined pre-beta- and beta-lipoproteins. Studies of fish eye disease patients and control subjects

The present study was undertaken to test our hypothesis that two different lecithin: cholesterol acyltransferase (LCAT) activities exist in normal human plasma, one denoted alpha-LCAT esterifying the free cholesterol of high density lipoproteins (HDL) and the other denoted beta-LCAT acting on the free cholesterol of very low (VLDL) and low (LDL) density lipoproteins. Plasmas depleted of HDL were obtained by means of preparative ultracentrifugation. Incubation at 37 degrees C of these plasma fractions from control subjects and patients with fish eye disease resulted in esterification of the remaining free cholesterol of combined VLDL and LDL (pre-beta- and beta-lipoproteins) in the HDL depleted plasmas. The shapes of the cholesterol esterification rate curves were similar for whole and HDL depleted plasmas from both control subjects and fish eye disease patients. In crosswise mixed incubation experiments with isolated combined VLD and LDL and total lipoprotein depleted plasma from a control subject and a patient with fish eye disease, respectively, esterification of free cholesterol occurred. Incubation of isolated total lipoproteins in plasma from a patient with LCAT deficiency mixed with total lipoprotein depleted plasma from a fish eye disease patient as a source of LCAT caused cholesterol esterification but did not result in normalization of the LCAT deficiency HDL particles, while the amount of normal-sized LDL particles increased. The present results support the hypothesis that a beta-LCAT exists in normal human plasma.     

Lipoprotein (a), low-density,intermediate-density lipoprotein,and blood pressure in a young male population

Summary Both hypercholesterolemia and hypertension are risk factors for atherosclerotic vascular disease, and elevated cholesterol levels occur more frequently than expected in patients with hypertension. Elevated levels of intermediate-density lipoproteins (IDL) and low-density lipoproteins (LDL) were shown to be atherogenic, and LDL, comprising the major cholesterol-carrying fraction in human plasma, are structurally related to lipoprotein (a) [Lp(a)], a further risk factor for atherosclerosis. In the present study we investigated 200 male employees (mean age 26±7 years) to determine whether the relationship of IDL and Lp(a) to systemic blood pressure is similar to the reported correlations between total and LDL cholesterol and systemic blood pressure. To this end blood pressure was measured several times in each individual, and lipids, lipoprotein-cholesterol, apolipoprotein B (apo B), and Lp(a) were determined in fasting serum. IDL cholesterol and apo B, the main protein component of IDL and LDL correlated with blood pressure. However, levels of Lp(a) correlated neither with systolic or diastolic blood pressure nor with lipoprotein cholesterol, body weight, or age. Although IDL and Lp(a) are considered lipoprotein risk factors for atherosclerosis, levels of Lp(a), unlike IDL, are not related to blood pressure, body weight, or age. Our data suggest different metabolic and pathophysiological mechanisms of the risk factors, IDL, LDL, and Lp(a).Abbreviations VLDL very low density lipoprotein - IDL intermediate-density lipoprotein - LDL low-density lipoprotein - ApoB Apolipoprotein B - Lp(a) lipoprotein (a) - BMI body mass index Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

Antibodies to oxidized LDL and LDL-containing immune complexes as risk factors for coronary artery disease in diabetes mellitus

Several groups have published results from clinical studies supporting the involvement of anti-modified LDL antibodies as risk factors for the initiation or progression of cardiovascular disease. However, the data published so far are judged inconclusive because of several contradictory observations concerning the correlation between clinical evidence of arteriosclerosis and the levels of antibodies to oxidized LDL (oxLDL Ab). We have previously reported that oxLDL Ab exist both in free form and as antigen-antibody complexes (LDL-IC) in patients with insulin-dependent diabetes mellitus (IDDM). The presence of LDL-IC in IDDM patients has important implications: it may interfere with the assay of oxLDL antibodies and the levels of LDL-IC may correlate better with the development of arteriosclerosis than the levels of free oxLDL antibodies. To clarify these questions baseline samples collected from 49 IDDM patients, who subsequently developed coronary artery disease (CAD) during an 8-year follow-up period, were compared to baseline samples from 49 age-, sex-, and duration-matched control IDDM subjects who remained free of clinical CAD during an identical follow-up period. The levels of free oxLDL antibody were significantly lower in the patients who developed CAD. The same patients had significantly higher concentrations of total cholesterol, apolipoprotein B, and IgA in immune complex-enriched polyethylene glycol (PEG) precipitates. The concentration of IgG was also higher in PEG precipitates from patients who developed CAD, but did not reach statistical significance. This indicates that patients who develop CAD had higher levels of circulating LDL-IC, a fact that could not be deduced from the measurement of free oxLDL antibody concentrations. A linear regression analysis of the correlation between the concentrations of total cholesterol in PEG precipitates, taken as a surrogate measurement of PEG-precipitated oxLDL-IC, and the concentration of free oxLDL antibody in serum showed a statistically significant negative correlation (r = -0.229, P = 0. 024). Our results support the conclusion that oxLDL-IC may be a risk factor for the development of macrovascular disease in IDDM patients. We also have demonstrated that circulating oxLDL-IC interfere with the assay of free oxLDL antibodies.     

Modified low density lipoproteins differentially bind and activate the C1 complex of complement

Several studies suggest that complement plays an important role in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of low density lipoprotein (LDL) to bind and activate C1, the complex that triggers the classical pathway of complement. For this purpose, LDL was both obtained commercially and purified according to an established procedure, and oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were generated from each preparation. Whereas the unmodified LDL and oxLDL samples did not activate C1 in the presence of excess C1 inhibitor, the E-LDL derivatives obtained by sequential treatment of LDL with a protease and then with cholesterol esterase triggered efficient C1 activation under these conditions, with activation levels approximately 60% upon incubation with 1 microM E-LDL for 90 min at 37 degrees C. In agreement with these findings, as shown by surface plasmon resonance spectroscopy (SPR), the C1q recognition subunit of C1 showed no interaction with unmodified LDL but bound to both E-LDL samples with high affinity (K(D)=58-75 nM). More unexpectedly, although they did not trigger direct C1 activation, both oxLDL samples were also efficiently recognized by C1q. Whereas the oxLDL derivative of commercial LDL activated C1 to a significant extent ( approximately 30%) in the presence of C-reactive protein (CRP), much lower activation levels (<10%) were obtained using the oxLDL derivative of purified LDL. As measured by SPR, CRP bound equally well to the oxLDL and E-LDL derivatives obtained from purified LDL. These data provide the first experimental evidence that E-LDL triggers efficient C1 activation under conditions close to the physiological situation, suggesting that activation of the classical complement pathway by this derivative may be a crucial factor in the pathogenesis of atherosclerosis. In contrast, it appears unlikely that oxLDL significantly activates C1 directly or in a CRP-dependent manner in vivo.     

Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe heritable hyperlipidemic rabbit.

Low density lipoproteins (LDL) that have been oxidatively modified have been implicated in the pathogenesis of atherosclerosis. Monoclonal antibodies were generated against oxidatively modified human low density lipoproteins (OxLDL); these antibodies reacted with OxLDL, but did not react with native LDL, either in an enzyme-linked immunosorbent assay (ELISA) or a Western blot analysis. The anti-OxLDL antibodies did react with other modified forms of LDL (eg, acetylated LDL, malondialdehyde-modified LDL, and cell-modified LDL) that were recognized by the scavenger receptor on macrophages. Single- and double-label immunofluorescence of atheromatous lesions from a Watanabe heritable hyperlipidemic (WHHL) rabbit demonstrated some colocalization of proteins detected by anti-LDL and anti-OxLDL antibodies. However, clearly there were also areas stained by the anti-OxLDL antibodies that did not stain with anti-LDL. Staining of the lesion by the anti-OxLDL antibody was abolished by adsorption of the antibody with OxLDL, but not by adsorption with LDL or bovine serum albumin. Arterial tissue from a control New Zealand White rabbit did not show staining with anti-LDL or anti-OxLDL antibodies. These observations suggest that OxLDL (or possibly other proteins recognized by the anti-OxLDL antibody) is present in atheromatous lesions of WHHL rabbits, and are consistent with oxidatively modified lipoproteins having a role in atherogenesis.