Heritability of Biomarkers of Oxidized Lipoproteins: Twin Pair Study.

OBJECTIVE: To determine whether biomarkers of oxidized lipoproteins are genetically determined. Lipoprotein(a) (Lp[a]) is a heritable risk factor and carrier of oxidized phospholipids (OxPL). APPROACH AND RESULTS: We measured oxidized phospholipids on apolipoprotein B-containing lipoproteins (OxPL-apoB), Lp(a), IgG, and IgM autoantibodies to malondialdehyde-modified low-density lipoprotein, copper oxidized low-density lipoprotein, and apoB-immune complexes in 386 monozygotic and dizygotic twins to estimate trait heritability (h(2)) and determine specific genetic effects among traits. A genome-wide linkage study followed by genetic association was performed. The h(2) (scale: 0-1) for Lp(a) was 0.91±0.01 and for OxPL-apoB 0.87±0.02, which were higher than physiological, inflammatory, or lipid traits. h(2) of IgM malondialdehyde-modified low-density lipoprotein, copper oxidized low-density lipoprotein, and apoB-immune complexes were 0.69±0.04, 0.67±0.05, and 0.80±0.03, respectively, and for IgG malondialdehyde-modified low-density lipoprotein, copper oxidized low-density lipoprotein, and apoB-immune complexes 0.62±0.05, 0.52±0.06, and 0.53±0.06, respectively. There was an inverse correlation between the major apo(a) isoform and OxPL-apoB (R=-0.49; P<0.001) and Lp(a) (R=-0.48; P<0.001) and OxPL-apoB was modestly correlated with Lp(a) (ρ=0.57; P<0.0001). The correlation in major apo(a) isoform size was concordant (R=1.0; P<0.001) among monozygotic twins but not dizygotic twins (R=0.40; P=0.055). Lp(a) and OxPL-apoB shared genetic codetermination (genetic covariance, ρG=0.774±0.032; P=1.09×10(-38)), although not environmental determination (environmental covariance, ρE=0.081±0.15; P=0.15). In contrast, Lp(a) shared environmental but not genetic codetermination with autoantibodies to malondialdehyde-modified low-density lipoprotein and copper oxidized low-density lipoprotein, and apoB-immune complexes. Sib-pair genetic linkage of the Lp(a) trait revealed that single nucleotide polymorphism rs10455872 was significantly associated with OxPL-apoB after adjusting for Lp(a). CONCLUSIONS: OxPL-apoB and other biomarkers of oxidized lipoproteins are highly heritable cardiovascular risk factors that suggest novel genetic origins of atherothrombosis.

Oxidized phospholipids are present on plasminogen, affect fibrinolysis, and increase following acute myocardial infarction.

OBJECTIVES: This study sought to assess whether plasminogen, which is homologous to lipoprotein (a) [Lp(a)], contains proinflammatory oxidized phospholipids (OxPL) and whether this has clinical relevance. BACKGROUND: OxPL measured on apolipoprotein B-100 (OxPL/apoB), primarily reflecting OxPL on Lp(a), independently predict cardiovascular disease (CVD) events. METHODS: The authors examined plasminogen from commercially available preparations and plasma from chimpanzees; gorillas; bonobos; cynomolgus monkeys; wild-type, apoE(-/-), LDLR(-/-), and Lp(a)-transgenic mice; healthy humans; and patients with familial hypercholesterolemia, stable CVD, and acute myocardial infarction (AMI). Phosphocholine (PC)-containing OxPL (OxPC) present on plasminogen were detected directly with liquid chromatography-mass spectrometry (LC-MS/MS) and immunologically with monoclonal antibody E06. In vitro clot lysis assays were performed to assess the effect of the OxPL on plasminogen on fibrinolysis. RESULTS: LC-MS/MS revealed that OxPC fragments were covalently bound to mouse plasminogen. Immunoblot, immunoprecipitation, density gradient ultracentrifugation, and enzyme-linked immunosorbent assay analyses demonstrated that all human and animal plasma samples tested contained OxPL covalently bound to plasminogen. In plasma samples subjected to density gradient fractionation, OxPL were present on plasminogen (OxPL/plasminogen) in non-lipoprotein fractions but on Lp(a) in lipoprotein fractions. Plasma levels of OxPL/apoB and OxPL/apo(a) varied significantly (>25×) among subjects and also strongly correlated with Lp(a) levels. In contrast, OxPL/plasminogen levels were distributed across a relatively narrow range and did not correlate with Lp(a). Enzymatic removal of OxPL from plasminogen resulted in a longer lysis time for fibrin clots (16.25 vs. 11.96 min, p = 0.007). In serial measurements over 7 months, OxPL/plasminogen levels did not vary in normal subjects or in patients with stable CVD, but increased acutely over the first month and then slowly decreased to baseline in patients following AMI. CONCLUSIONS: These data demonstrate that plasminogen contains covalently bound OxPL that influence fibrinolysis. OxPL/plasminogen represent a second major plasma pool of OxPL, in addition to those present on Lp(a). OxPL present on plasminogen may have pathophysiological implications in AMI and atherothrombosis.

Relationship of IgG and IgM autoantibodies and immune complexes to oxidized LDL with markers of oxidation and inflammation and cardiovascular events: results from the EPIC-Norfolk Study.

Levels of IgG and IgM autoantibodies (AA) to malondialdehyde (MDA)-LDL and apoB-immune complexes (ICs) were measured in 748 cases and 1,723 controls in the EPIC-Norfolk cohort and their association to coronary artery disease (CAD) events determined. We evaluated whether AA and IC modify CAD risk associated with secretory phospholipase A(2) (sPLA(2)) type IIA mass and activity, lipoprotein-associated PLA(2) activity, lipoprotein (a) [Lp(a)], oxidized phospholipids on apoB-100 (OxPL/apoB), myeloperoxidase, and high sensitivity C-reactive protein. IgG ICs were higher in cases versus controls (P = 0.02). Elevated levels of IgM AA and IC were inversely associated with Framingham Risk Score and number of metabolic syndrome criteria (p range 0.02-0.001). In regression analyses adjusted for age, smoking, diabetes, LDL-cholesterol, HDL-cholesterol, and systolic blood pressure, the highest tertiles of IgG and IgM AA and IC were not associated with higher risk of CAD events compared with the lowest tertiles. However, elevated levels of IgM IC reduced the risk of Lp(a) (P = 0.006) and elevated IgG MDA-LDL potentiated the risk of sPLA(2) mass (P = 0.018). This epidemiological cohort of initially healthy subjects shows that IgG and IgM AA and IC are not independent predictors of CAD events but may modify CAD risk associated with elevated levels of oxidative biomarkers.

Antisense oligonucleotide lowers plasma levels of apolipoprotein (a) and lipoprotein (a) in transgenic mice.

OBJECTIVES: This study sought to assess whether an antisense oligonucleotide (ASO) directed to apolipoprotein (a) [apo(a)] reduces apo(a) and lipoprotein (a) [Lp(a)] levels in transgenic mouse models. BACKGROUND: Elevated Lp(a) is a causal, independent, genetic risk factor for cardiovascular disease and myocardial infarction. Effective therapies to specifically lower plasma Lp(a) levels are lacking. METHODS: Three transgenic mouse models were utilized: 8K-apo(a) mice expressing 8 kringle IV (KIV) repeats with a single copy of KIV-2; 8K-Lp(a) mice expressing both the 8K apo(a) plus human apolipoprotein B-100; and 12K-apo(a) mice expressing a 12K apo(a) with 3 KIV-2 repeats. The mice were treated intraperitoneally with saline, a control ASO, or ASO 144367 directed to KIV-2 for 4 to 6 weeks. Apo(a), Lp(a), and oxidized phospholipids present on human apoB (OxPL/h-apoB) or apo(a) [OxPL/apo(a)] were measured at baseline and on and off therapy. RESULTS: ASO 144367 significantly reduced Lp(a) by 24.8% in 8K-Lp(a) mice, and reduced apo(a) levels by 19.2% in 8K-Lp(a) mice, 30.0% in 8K-apo(a) mice, and 86% in 12K-apo(a) mice; ASO 144367 also significantly reduced OxPL/apoB 22.4% in 8K-Lp(a) mice, and OxPL/apo(a) levels by 19.9% in 8K-Lp(a) mice, 22.1% in 8K-apo(a) mice, and 92.5% in 12K-apo(a) mice (p < 0.004, or less, for all). No significant changes occurred in Lp(a), apo(a), OxPL/apoB, or OxPL/apo(a) levels with control ASO or saline. CONCLUSIONS: This study documents the first specific therapy, to our knowledge, for lowering apo(a)/Lp(a) levels and their associated OxPL. A more potent effect was documented in mice expressing apo(a) with multiple KIV-2 repeats. Targeting liver expression of apo(a) with ASOs directed to KIV-2 repeats may provide an effective approach to lower elevated Lp(a) levels in humans.

Oxidation-specific biomarkers, lipoprotein(a), and risk of fatal and nonfatal coronary events.

OBJECTIVES: This study sought to assess whether oxidation-specific biomarkers are associated with an increased risk of coronary artery disease (CAD) events. BACKGROUND: The relationship of a panel of oxidative biomarkers and lipoprotein(a) [Lp(a)] to CAD risk is not fully determined. METHODS: A prospective case-control study nested in the EPIC (European Prospective Investigation of Cancer)-Norfolk cohort of 45- to 79-year-old apparently healthy men and women followed for approximately 6 years was designed. Cases consisted of participants in whom fatal or nonfatal CAD developed, matched by sex, age, and enrollment time with controls without CAD. Baseline levels of oxidized phospholipids on apolipoprotein B-100 particles and Lp(a) were measured in 763 cases and 1,397 controls. Their relationship to secretory phospholipase A(2) type IIA mass and activity, myeloperoxidase mass, and lipoprotein-associated phospholipase A(2) activity and association with CAD events were determined. RESULTS: After adjusting for age, smoking, diabetes, low- and high-density lipoprotein cholesterol, and systolic blood pressure, the highest tertiles of oxidized phospholipids on apolipoprotein B-100 particles and Lp(a) were associated with a significantly higher risk of CAD events (odds ratios: 1.67 and 1.64, respectively; p < 0.001) compared with the lowest tertiles. The odds ratio of CAD events associated with the highest tertiles of oxidized phospholipids on apolipoprotein B-100 particles or Lp(a) was significantly potentiated (approximately doubled) by the highest tertiles of secretory phospholipase A(2) activity and mass but less so for myeloperoxidase and lipoprotein-associated phospholipase A(2) activity. The odds ratios for fatal CAD were higher than for the combined end point. After taking into account the Framingham Risk Score, c-index values progressively increased when oxidative biomarkers were added to the model. CONCLUSIONS: This EPIC-Norfolk study links pathophysiologically related oxidation-specific biomarkers and Lp(a) with CAD events. Oxidation-specific biomarkers provide cumulative predictive value when added to traditional cardiovascular risk factors.

Changes in lipoprotein(a), oxidized phospholipids, and LDL subclasses with a low-fat high-carbohydrate diet.

Low-fat diets have been shown to increase plasma concentrations of lipoprotein(a) [Lp(a)], a preferential lipoprotein carrier of oxidized phospholipids (OxPLs) in plasma, as well as small dense LDL particles. We sought to determine whether increases in plasma Lp(a) induced by a low-fat high-carbohydrate (LFHC) diet are related to changes in OxPL and LDL subclasses. We studied 63 healthy subjects after 4 weeks of consuming, in random order, a high-fat low-carbohydrate (HFLC) diet and a LFHC diet. Plasma concentrations of Lp(a) (P < 0.01), OxPL/apolipoprotein (apo)B (P < 0.005), and OxPL-apo(a) (P < 0.05) were significantly higher on the LFHC diet compared with the HFLC diet whereas LDL peak particle size was significantly smaller (P < 0.0001). Diet-induced changes in Lp(a) were strongly correlated with changes in OxPL/apoB (P < 0.0001). The increases in plasma Lp(a) levels after the LFHC diet were also correlated with decreases in medium LDL particles (P < 0.01) and increases in very small LDL particles (P < 0.05). These results demonstrate that induction of increased levels of Lp(a) by an LFHC diet is associated with increases in OxPLs and with changes in LDL subclass distribution that may reflect altered metabolism of Lp(a) particles.

Inhibition of arterial lesion progression in CD16-deficient mice: evidence for altered immunity and the role of IL-10.

AIMS: Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR(-/-)) and FcgRIII(-/-)xLDLR(-/-) double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet. METHODS AND RESULTS: Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR(-/-) controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR(-/-) controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-gamma, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII (-/-)xLDLR(-/-) double knockouts relative to LDLR(-/-) controls, while total IgG levels were similar. CONCLUSION: These results reveal altered immunity in FcgRIII(-/-)xLDLR(-/-) mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.

The I4399M variant of apolipoprotein(a) is associated with increased oxidized phospholipids on apolipoprotein B-100 particles.

OBJECTIVES: The LPA I4399M (rs3798220) single nucleotide polymorphism (SNP) is associated with increased plasma levels of Lp(a) and advanced coronary artery disease (CAD). We hypothesized that carriers of the Met allele of the I4399M SNP would also have elevated levels of oxidized phospholipids (OxPL) on apoB (OxPL/apoB) particles. METHODS AND RESULTS: Plasma levels of Lp(a) and OxPL/apoB were measured in non-carriers (TT genotype, n=116) and carriers (CT/CC genotype, n=58) of the I4399M SNP. Carriers had significantly higher Lp(a) levels [median (interquartile range, IQR)] [43 (9-57)mg/dl vs. 5.5 (2-20)mg/dl, p<0.0001] and smaller apolipoprotein(a) isoforms than non-carriers (number of kringle IV repeats: 18(17-20) vs. 22(18-25), p=0.002). Median (IQR) OxPL/apoB levels were significantly higher in carriers than non-carriers [8019 (6254-31,785) relative light units (RLU) vs. 2168 (1303-5869), p<0.0001]. Patients with small apolipoprotein(a) isoforms had the highest OxPL/apoB levels. The number of kringle IV repeats was inversely related to Lp(a) (r=-0.43, p<0.001) and OxPL/apoB (r=-0.36, p<0.0001) levels. CONCLUSION: The CT and CC genotypes of the I4399M SNP in the LPA gene are associated with elevated OxPL/apoB levels, which primarily represent OxPL on Lp(a). The concomitant increase of OxPL/apoB levels in the setting of small apolipoprotein(a) isoforms may potentiate the atherogenic effect on CAD of elevated Lp(a) levels in carriers of the I4399M SNP.

Conventional dendritic cells at the crossroads between immunity and cholesterol homeostasis in atherosclerosis.

BACKGROUND: Immunoinflammatory mechanisms are implicated in the atherogenic process. The polarization of the immune response and the nature of the immune cells involved, however, are major determinants of the net effect, which may be either proatherogenic or antiatherogenic. Dendritic cells (DCs) are central to the regulation of immunity, the polarization of the immune response, and the induction of tolerance to antigens. The potential role of DCs in atherosclerosis, however, remains to be defined. METHODS AND RESULTS: We created a mouse model in which the lifespan and immunogenicity of conventional DCs are enhanced by specific overexpression of the antiapoptotic gene hBcl-2 under the control of the CD11c promoter. When studied in either low-density lipoprotein receptor-deficient or apolipoprotein E-deficient backgrounds, DC-hBcl2 mice exhibited an expanded DC population associated with enhanced T-cell activation, a T-helper 1 and T-helper 17 cytokine expression profile, and elevated production of T-helper 1-driven IgG2c autoantibodies directed against oxidation-specific epitopes. This proatherogenic signature, however, was not associated with acceleration of atherosclerotic plaque progression, because expansion of the DC population was unexpectedly associated with an atheroprotective decrease in plasma cholesterol levels. Conversely, depletion of DCs in hyperlipidemic CD11c-diphtheria toxin receptor/apolipoprotein E-deficient transgenic mice resulted in enhanced cholesterolemia, thereby arguing for a close relationship between the DC population and plasma cholesterol levels. CONCLUSIONS: Considered together, the present data reveal that conventional DCs are central to the atherosclerotic process, because they are directly implicated in both cholesterol homeostasis and the immune response.

Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.

Lipid accumulation in arteries induces vascular inflammation and atherosclerosis, the major cause of heart attack and stroke in humans. Extreme hyperlipidemia induced in mice and rabbits enables modeling many aspects of human atherosclerosis, but microscopic examination of plaques is possible only postmortem. Here we report that feeding adult zebrafish (Danio rerio) a high-cholesterol diet (HCD) resulted in hypercholesterolemia, remarkable lipoprotein oxidation, and fatty streak formation in the arteries. Feeding an HCD supplemented with a fluorescent cholesteryl ester to optically transparent fli1:EGFP zebrafish larvae in which endothelial cells express green fluorescent protein (GFP), and using confocal microscopy enabled monitoring vascular lipid accumulation and the endothelial cell layer disorganization and thickening in a live animal. The HCD feeding also increased leakage of a fluorescent dextran from the blood vessels. Administering ezetimibe significantly diminished the HCD-induced endothelial cell layer thickening and improved its barrier function. Feeding HCD to lyz:DsRed2 larvae in which macrophages and granulocytes express DsRed resulted in the accumulation of fluorescent myeloid cells in the vascular wall. Using a fluorogenic substrate for phospholipase A(2) (PLA(2)), we observed an increased vascular PLA(2) activity in live HCD-fed larvae compared to control larvae. Furthermore, by transplanting genetically modified murine cells into HCD-fed larvae, we demonstrated that toll-like receptor-4 was required for efficient in vivo lipid uptake by macrophages. These results suggest that the novel zebrafish model is suitable for studying temporal characteristics of certain inflammatory processes of early atherogenesis and the in vivo function of vascular cells.

Relationship of oxidized phospholipids on apolipoprotein B-100 particles to race/ethnicity, apolipoprotein(a) isoform size, and cardiovascular risk factors: results from the Dallas Heart Study.

BACKGROUND: Elevated levels of oxidized phospholipids (OxPLs) on apolipoprotein B-100 particles (OxPL/apoB) are associated with cardiovascular disease and predict new cardiovascular events. Elevated lipoprotein (a) [Lp(a)] levels are a risk factor for cardiovascular disease in whites and also in blacks if they carry small apolipoprotein(a) [apo(a)] isoforms. The relationship of OxPL/apoB levels to race/ethnicity, cardiovascular risk factors, and apo(a) isoforms is not established. METHODS AND RESULTS: OxPL/apoB levels were measured in 3481 subjects (1831 black, 1047 white, and 603 Hispanic subjects) in the Dallas Heart Study and correlated with age, sex, cardiovascular risk factors, and Lp(a) and apo(a) isoforms. Significant differences in OxPL/apoB levels were noted among racial/ethnic subgroups, with blacks having the highest levels compared with whites and Hispanics (P<0.001 for each comparison). OxPL/apoB levels generally did not correlate with age, sex, or risk factors. In the overall cohort, OxPL/apoB levels strongly correlated with Lp(a) (r=0.85, P<0.001), with the shape of the relationship demonstrating a "reverse L" shape for log-transformed values. The highest correlation was present in blacks, followed by whites and Hispanics; was dependent on apo(a) isoform size; and became progressively weaker with larger isoforms. The size of the major apo(a) isoform (number of kringle type IV repeats) was negatively associated with OxPL/apoB (r=-0.49, P<0.001) and Lp(a) (r=-0.61, P<0.001) regardless of racial/ethnic group. After adjustment for apo(a) isoform size, the relationship between OxPL/apoB and Lp(a) remained significant (r=0.67, P<0.001). CONCLUSIONS: OxPL/apoB levels vary according to race/ethnicity, are largely independent of cardiovascular risk factors, and are inversely associated with apo(a) isoform size. The association of OxPL with small apo(a) isoforms, in which a similar relationship is present among all racial/ethnic subgroups despite differences in Lp(a) levels, may be a key determinant of cardiovascular risk.

Macrophage apoptosis exerts divergent effects on atherogenesis as a function of lesion stage.

BACKGROUND: Because apoptotic cell clearance appears to be defective in advanced compared with early atherosclerotic plaques, macrophage apoptosis may differentially affect plaque progression as a function of lesion stage. METHODS AND RESULTS: We first evaluated the impact of targeted protection of macrophages against apoptosis at both early and advanced stages of atherosclerosis. Increased resistance of macrophages to apoptosis in early atherosclerotic lesions was associated with increased plaque burden; in contrast, it afforded protection against progression to advanced lesions. Conversely, sustained induction of apoptosis in lesional macrophages of advanced lesions resulted in a significant increase in lesion size. Such enhanced lesion size occurred as a result not only of apoptotic cell accumulation but also of elevated chemokine expression and subsequent intimal recruitment of circulating monocytes. CONCLUSIONS: Considered together, our data suggest that macrophage apoptosis is atheroprotective in fatty streak lesions, but in contrast, defective clearance of apoptotic debris in advanced lesions favors arterial wall inflammation and enhanced recruitment of monocytes, leading to enhanced atherogenesis.

A novel function of lipoprotein [a] as a preferential carrier of oxidized phospholipids in human plasma.

Oxidized phospholipids (OxPLs) on apolipoprotein B-100 (apoB-100) particles are strongly associated with lipoprotein [a] (Lp[a]). In this study, we evaluated whether Lp[a] is preferentially the carrier of OxPL in human plasma. The content of OxPL on apoB-100 particles was measured with monoclonal antibody E06, which recognizes the phosphocholine (PC) headgroup of oxidized but not native phospholipids. To assess whether OxPLs were preferentially bound by Lp[a] as opposed to other lipoproteins, immunoprecipitation and ultracentrifugation experiments, in vitro transfer studies, and chemiluminescent ELISAs were performed. Immunoprecipitation of Lp[a] from human plasma with an apolipoprotein [a] (apo[a])-specific antibody demonstrated that more than 85% of E06 reactivity (i.e., OxPL) coimmunoprecipitated with Lp[a]. Ultracentrifugation experiments showed that nearly all OxPLs were found in fractions containing apo[a], as opposed to other apolipoproteins. In vitro transfer studies showed that oxidized LDL preferentially donates OxPLs to Lp[a], as opposed to LDL, in a time- and temperature-dependent manner, even in aqueous buffer. Approximately 50% of E06 immunoreactivity could be extracted from isolated Lp[a] following exposure of plasma to various lipid solvents. These data demonstrate that Lp[a] is the preferential carrier of PC-containing OxPL in human plasma. This unique property of Lp[a] suggests novel insights into its physiological function and mechanisms of atherogenicity.

Antisense oligonucleotide directed to human apolipoprotein B-100 reduces lipoprotein(a) levels and oxidized phospholipids on human apolipoprotein B-100 particles in lipoprotein(a) transgenic mice.

BACKGROUND: Lipoprotein (a) [Lp(a)] is a genetic cardiovascular risk factor that preferentially binds oxidized phospholipids (OxPL) in plasma. There is a lack of therapeutic agents that reduce plasma Lp(a) levels. METHODS AND RESULTS: Transgenic mice overexpressing human apolipoprotein B-100 (h-apoB-100 [h-apoB mice]) or h-apoB-100 plus human apo(a) to generate genuine Lp(a) particles [Lp(a) mice] were treated with the antisense oligonucleotide mipomersen directed to h-apoB-100 mRNA or control antisense oligonucleotide for 11 weeks by intraperitoneal injection. Mice were then followed up for an additional 10 weeks off therapy. Lp(a) levels [apo(a) bound to apoB-100] and apo(a) levels ["free" apo(a) plus apo(a) bound to apoB-100] were measured by chemiluminescent enzyme-linked immunoassay and commercial assays, respectively. The content of OxPL on h-apoB-100 particles (OxPL/h-apoB) was measured by capturing h-apoB-100 in microtiter wells and detecting OxPL by antibody E06. As expected, mipomersen significantly reduced plasma h-apoB-100 levels in both groups of mice. In Lp(a) mice, mipomersen significantly reduced Lp(a) levels by approximately 75% compared with baseline (P<0.0001) but had no effect on apo(a) levels or hepatic apo(a) mRNA expression. OxPL/h-apoB levels were much higher at baseline in Lp(a) mice compared with h-ApoB mice (P<0.0001) but decreased in a time-dependent fashion with mipomersen. There was no effect of the control antisense oligonucleotide on lipoprotein levels or oxidative parameters. CONCLUSIONS: Mipomersen significantly reduced Lp(a) and OxPL/apoB levels in Lp(a) mice. The present study demonstrates that h-apoB-100 is a limiting factor in Lp(a) particle synthesis in this Lp(a) transgenic model. If applicable to humans, mipomersen may represent a novel therapeutic approach to reducing Lp(a) levels and their associated OxPL.

Oxidized low-density lipoprotein biomarkers in patients with end-stage renal failure: acute effects of hemodialysis.

OBJECTIVE: To assess the effect of end-stage renal failure on oxidized low-density lipoprotein (OxLDL) biomarkers and the acute effects of hemodialysis. Oxidized phospholipids (OxPL) on apolipoprotein B-100 (apoB) particles (OxPL/apoB) have been associated with cardiovascular disease and new cardiovascular events. Patients with end-stage renal failure have increased oxidative stress and are at significantly increased risk of cardiovascular disease. METHODS AND RESULTS: Fifty-two stable patients with end-stage renal failure undergoing chronic hemodialysis were included in the study. Pre and post hemodialysis blood samples were obtained for measurement of OxLDL biomarkers: oxidized phospholipids (OxPL) on apolipoprotein B-100 (apoB) particles (OxPL/apoB) measured by antibody E06, IgG and IgM autoantibody titers to copper-oxidized LDL (Cu-OxLDL) and malondialdehyde (MDA)-LDL, IgG and IgM apolipoprotein B-100-immune complexes (IC/apoB). Traditional laboratory variables as well as C-reactive protein (CRP) and lipoprotein(a) [Lp(a)] were also measured. For the baseline variables, the distribution of OxPL/apoB and Lp(a) were skewed to lower values, and a strong correlation was noted between OxPL/apoB and Lp(a) (r = 0.94, p < 0.0001). No major associations were noted between OxLDL biomarkers and age, gender or dialytic age. There were also no correlations between OxLDL biomarkers and traditional risk factors, CRP, body mass index, serum creatinine, hypertension or intravenous iron therapy. Following dialysis, there as a significant reduction in OxPL/apoB (-7.5%, p = 0.048) and triglyceride levels (-10.8%, p = 0.005). All other OxLDL biomarkers, CRP, total cholesterol, LDL-C, HDL-C and apoB-100 increased significantly (range 6.3-26.9%, p value range 0.005 to <0.0001). Total protein plasma levels increased 8.8% (p = 0.014 compared to predialysis) following dialysis, consistent with a hemoconcentration effect of hemodialysis. CONCLUSION: In end-stage renal failure patients undergoing hemodialysis, a reduction in OxPL/apoB levels was noted, despite the hemoconcentrating and strong pro-oxidant milieu of hemodialysis. Studies in larger populations of end-stage renal failure patients are needed to assess whether these findings predict future clinical outcomes.

Relationship of IgG and IgM autoantibodies to oxidized low density lipoprotein with coronary artery disease and cardiovascular events.

The relationship between autoantibodies to oxidized low density lipoprotein (OxLDL) and coronary artery disease (CAD) remains controversial. IgM and IgG OxLDL autoantibodies to malondialdehyde (MDA)-modified LDL, copper oxidized low density lipoprotein (CuOxLDL), and oxidized cholesterol linoleate (OxCL), as well as apolipoprotein B-100 immune complexes (apoB-ICs), were measured in 504 patients undergoing clinically indicated coronary angiography. Patients were followed for cardiovascular events for a median of 4 years. In univariate analysis, IgM OxLDL autoantibodies and IgM apoB-ICs were inversely associated with the presence of angiographically determined CAD, whereas IgG OxLDL autoantibodies and IgG apoB-ICs were positively associated. In logistic regression analysis, compared with the first quartile, patients in the fourth quartile of IgM OxLDL autoantibodies and apoB-ICs showed a lower probability of angiographically determined CAD (>50% diameter stenosis). Odds ratios and (95% confidence intervals) were as follows: MDA-LDL, 0.51 (0.32-0.82; P = 0.005); CuOxLDL, 0.63 (0.39-1.01; P = 0.05); OxCL, 0.63 (0.39-1.01; P = 0.05); and apoB-IC, 0.55 (0.34-0.88; P = 0.013). These relationships were accentuated in the setting of hypercholesterolemia, with the highest IgM levels showing the lowest risk of CAD for the same level of hypercholesterolemia. Multivariable analysis revealed that neither IgM or IgG OxLDL autoantibodies nor apoB-ICs were independently associated with angiographically determined CAD or cardiovascular events. In conclusion, IgG and IgM OxLDL biomarkers have divergent associations with CAD in univariate analysis but are not independent predictors of CAD or clinical events.

Increased plasma oxidized phospholipid:apolipoprotein B-100 ratio with concomitant depletion of oxidized phospholipids from atherosclerotic lesions after dietary lipid-lowering: a potential biomarker of early atherosclerosis regression.

BACKGROUND: Oxidized phospholipids (OxPL) are pro-inflammatory. We evaluated whether changes in plasma levels of OxPL associated with apolipoprotein B-100 (apoB-100) reflect changes in OxPL content in atherosclerotic plaques during dietary-induced atherosclerosis progression and regression. METHODS AND RESULTS: OxPL content was measured in plasma and immunohistochemically in aortic plaques with antibody E06 in cynomolgus monkeys and New Zealand White rabbits at baseline, after a high-fat/high-cholesterol diet and after reversion to normal chow. The OxPL/apoB ratio, representing the content of OxPL on individual apoB-100 particles, and Total apoB-OxPL (OxPL/apoB multiplied by plasma apoB levels), reflecting the OxPL content on all apoB-100 particles, were measured. Total apoB-OxPL plasma levels increased 3-fold (P<0.0001) during hypercholesterolemia and decreased approximately 75% (P<0.0001) during reversion to normocholesterolemia. In contrast, OxPL/apoB levels decreased significantly (P<0.0001) during hypercholesterolemia and increased significantly (P=0.0002) during reversion to normocholesterolemia. Immunostaining revealed that during atherosclerosis progression OxPL co-localized with apoB-100, whereas during regression OxPL virtually disappeared. CONCLUSIONS: In the setting of overall reduction of plasma OxPL levels after dietary lipid-lowering, increases in the OxPL/apoB ratio reflect reduced content of OxPL in atherosclerotic plaques. These data suggest that changes in the OxPL/apoB ratio may reflect early atherosclerosis regression.

Oxidized phospholipids predict the presence and progression of carotid and femoral atherosclerosis and symptomatic cardiovascular disease: five-year prospective results from the Bruneck study.

OBJECTIVES: The purpose of this work was to determine the predictive value of oxidized phospholipids (OxPLs) present on apolipoprotein B-100 particles (apoB) in carotid and femoral atherosclerosis. BACKGROUND: The OxPLs are pro-inflammatory and pro-atherogenic and may be detected using the antibody E06 (OxPL/apoB). METHODS: The Bruneck study is a prospective population-based survey of 40- to 79-year-old men and women initiated in 1990. Plasma levels of OxPL/apoB and lipoprotein (a) [Lp(a)] were measured in 765 of 826 (92.6%) and 671 of 684 (98.1%) subjects alive in 1995 and 2000, respectively, and correlated with ultrasound measures of carotid and femoral atherosclerosis. RESULTS: The distribution of the OxPL/apoB levels was skewed to lower levels and nearly identical to Lp(a) levels. The OxPL/apoB and Lp(a) levels were highly correlated (r = 0.87, p < 0.001), and displayed long-term stability and lacked correlations with most cardiovascular risk factors and lifestyle variables. The number of apolipoprotein (a) kringle IV-2 repeats was inversely related to Lp(a) mass (r = -0.48, p < 0.001) and OxPL/apoB levels (r = -0.46, p < 0.001). In multivariable analysis, OxPL/apoB levels were strongly and significantly associated with the presence, extent, and development (1995 to 2000) of carotid and femoral atherosclerosis and predicted the presence of symptomatic cardiovascular disease. Both OxPL/apoB and Lp(a) levels showed similar associations with atherosclerosis severity and progression, suggesting a common biological influence on atherogenesis. CONCLUSIONS: This study suggests that pro-inflammatory oxidized phospholipids, present primarily on Lp(a), are significant predictors of the presence and extent of carotid and femoral atherosclerosis, development of new lesions, and increased risk of cardiovascular events. The OxPL biomarkers may provide valuable insights into diagnosing and monitoring cardiovascular disease.

Oxidized low-density lipoprotein in children with familial hypercholesterolemia and unaffected siblings: effect of pravastatin.

OBJECTIVES: To assess the role of oxidized phospholipids (OxPLs) in children with familial hypercholesterolemia (FH) and the effect of pravastatin. BACKGROUND: Oxidized phospholipids are a major component of oxidized low-density lipoprotein (OxLDL) and are bound to lipoprotein (a) [Lp(a)]. The significance of OxPL markers in children is unknown. METHODS: Children with FH were randomized to placebo (n = 88) or pravastatin (n = 90) after instruction on American Heart Association step II diet. Unaffected siblings (n = 78) served as controls. The OxPL content on apolipoprotein B-100 (apoB) detected by antibody E06 (OxPL/apoB ratio), immunoglobulin (Ig)G and IgM immune complexes per apoB (IC/apoB) and on all apoB particles (total apoB-IC = IC/apoB multiplied by plasma apoB levels), autoantibodies to malondialdehyde (MDA)-low-density lipoprotein (LDL), Lp(a), and apoB levels were measured at baseline and after two years of treatment. RESULTS: Compared with unaffected siblings, children with FH had significantly lower levels of OxPL/apoB but higher levels of IgG and IgM total apoB-IC and IgM MDA-LDL autoantibodies. From baseline to two-year follow-up, compared with placebo pravastatin treatment resulted in a greater mean percentage change in apoB (-18.7% vs. 0.3%; p = 0.001), total IgG apoB-IC (-31.9% vs. -12.2%; p < 0.001), and total IgM apoB-IC (-25.5% vs. 13.2%; p = 0.001). Interestingly, pravastatin also resulted in higher OxPL/apoB (48.7% vs. 29.3%; p = 0.028) and Lp(a) levels (21.9% vs. 10.7%; p = 0.044). CONCLUSIONS: Compared with unaffected siblings, children with FH are characterized by elevated levels of apoB-IC and IgM MDA-LDL autoantibodies. Compared with placebo, pravastatin led to a greater reduction in apoB-IC but also to a greater increase in OxPL/apoB and Lp(a), which may represent a novel mechanism of mobilization and clearance of OxPL.

Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease.

BACKGROUND: Lp(a) lipoprotein binds proinflammatory oxidized phospholipids. We investigated whether levels of oxidized low-density lipoprotein (LDL) measured with use of monoclonal antibody E06 reflect the presence and extent of obstructive coronary artery disease, defined as a stenosis of more than 50 percent of the luminal diameter. METHODS: Levels of oxidized LDL and Lp(a) lipoprotein were measured in a total of 504 patients immediately before coronary angiography. Levels of oxidized LDL are reported as the oxidized phospholipid content per particle of apolipoprotein B-100 (oxidized phospholipid:apo B-100 ratio). RESULTS: Measurements of the oxidized phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels were skewed toward lower values, and the values for the oxidized phospholipid:apo B-100 ratio correlated strongly with those for Lp(a) lipoprotein (r=0.83, P<0.001). In the entire cohort, the oxidized phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels showed a strong and graded association with the presence and extent of coronary artery disease (i.e., the number of vessels with a stenosis of more than 50 percent of the luminal diameter) (P<0.001). Among patients 60 years of age or younger, those in the highest quartiles for the oxidized phospholipid:apo B-100 ratio and Lp(a) lipoprotein levels had odds ratios for coronary artery disease of 3.12 (P<0.001) and 3.64 (P<0.001), respectively, as compared with patients in the lowest quartile. The combined effect of hypercholesterolemia and being in the highest quartiles of the oxidized phospholipid:apo B-100 ratio (odds ratio, 16.8; P<0.001) and Lp(a) lipoprotein levels (odds ratio, 14.2; P<0.001) significantly increased the probability of coronary artery disease among patients 60 years of age or younger. In the entire study group, the association of the oxidized phospholipid:apo B-100 ratio with obstructive coronary artery disease was independent of all clinical and lipid measures except one, Lp(a) lipoprotein. However, among patients 60 years of age or younger, the oxidized phospholipid:apo B-100 ratio remained an independent predictor of coronary artery disease. CONCLUSIONS: Circulating levels of oxidized LDL are strongly associated with angiographically documented coronary artery disease, particularly in patients 60 years of age or younger. These data suggest that the atherogenicity of Lp(a) lipoprotein may be mediated in part by associated proinflammatory oxidized phospholipids.