Oxidized phospholipids on apoB-100-containing lipoproteins: a biomarker predicting cardiovascular disease and cardiovascular events

Abstract

Oxidative stress is a well-known etiologic factor in the development of cardiovascular disease. Oxidation of lipoproteins, and in particular of low density lipoprotein, is a necessary if not obligatory mechanism for the generation of macrophage-derived foam cells, the first major initiating factor in the development of an atherosclerotic plaque. Oxidation of lipoproteins does not result in the generation of a single, defined molecular species, but of a variety of oxidation-specific epitopes, such as oxidized phospholipids and malondialdehyde-lysine epitopes. Unique monoclonal antibodies have been developed to bind these well-defined epitopes, and have been used in in vitro assays to detect them on circulating lipoproteins present in plasma. This article will summarize the accumulating clinical data of one oxidation-specific biomarker, oxidized phospholipids (OxPL) on apoB-100 lipoproteins. Elevated levels of OxPL/apoB predict the presence and progression of coronary, femoral and carotid artery disease, are increased following acute coronary syndromes and percutaneous coronary intervention, and predict the development of death, myocardial infarction, stroke and need for revascularization in unselected populations. OxPL/apoB levels are independent of traditional risk factors and the metabolic syndrome, and enhance the risk prediction of the Framingham Risk Score. The OxPLs measured in this assay reflect the biological activity of the most atherogenic lipoprotein(a) (Lp(a)) particles, reflected in patients with high plasma Lp(a) levels with small apo(a) isoforms. The predictive value of OxPL/apoB is amplified by Lp(a) and phospholipases such as lipoprotein-associated phospholipase A(2) and secretory phospholipase A(2), which are targets of therapy in clinical trials. This assay has now been validated in over 10,000 patients and efforts are underway to make it available to the research and clinical communities.

Figure 1. Oxidized lipid moieties induce lipoprotein accumulation in macrophages

Macrophage lipoprotein uptake mechanisms can be separated into: macropinocytosis, when actin polymerization and extensive membrane ruffling result in the ruffles closing into large endosomes and capture of large volumes of extracellular material, including all classes of native and OxLDL present in the vicinity of the cell; and micropinocytosis, when ligand–receptor binding leads to membrane invagination and nearly stoichiometric internalization of the ligand or the lipoprotein carrying this ligand. mmLDL and polyoxygenated OxCEs induce Syk recruitment to TLR-4, Syk and TLR-4 phosphorylation and subsequent ERK1/2-dependent activation of small GTPases Rac, cdc42 and Rho and phosphorylation of paxillin, leading to actin reorganization and membrane ruffling. Resulting macropinocytosis promotes foam cell formation [74]. Binding of OxLDL or OxPL to CD36 initiates Lyn-dependent phosphorylation of JNK, which is essential for CD36-mediated OxLDL uptake, although the mechanism linking JNK with the membrane dynamics is unclear [75]. The TLR-4- and CD36-mediated uptake mechanisms are only examples; there are numerous other pattern recognition receptors involved in oxidation-specific epitope-stimulated lipoprotein internalization by macrophages. mmLDL: Minimally modified low-density lipoprotein; nLDL: Native low-density lipoprotein; OxCE: Oxidized cholesterol ester; OxLDL: Oxidized low-density lipoprotein; OxPL: Oxidized phospholipid; POVPC: 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine; TLR: Toll-like receptor. Adapted with permission from [26].

Figure 2. Pattern recognition of oxidation-specific danger-associated molecular patterns and microbial pathogen-associated molecular patterns

Using the example of the PC epitope, in this illustration, we demonstrate our hypothesis of the emergence and positive selection of multiple pattern recognition receptors (PRRs) that recognize common epitopes, shared by modified self- and microbial pathogens. According to this hypothesis, oxidation of plasma membrane phospholipids in apoptotic cells alters the conformation of the PC head group, yielding an exposed epitope, accessible to recognition by macrophage scavenger receptors, natural antibodies and pentraxins, such as C-reactive protein. These PRRs were selected to clear apoptotic cells from developing or regenerating tissues. Recognition by the same receptors of the PC epitope of capsular polysaccharide in Gram-positive bacteria (e.g., Streptococcus pneumoniae) strengthened positive selection of these PRRs and probably helped to select additional strong proinflammatory components to PRR-dependent responses. (Note that the PC on the bacteria is not part of a phospholipid.) Finally, oxidized lipoproteins, prevalent in experimental animals and humans as a result of enhanced oxidative stress, dyslipidemia and impact of environmental factors, bear OxPLs with the PC epitope exposed in an analogous manner to that of apoptotic cells. This leads to OxLDL recognition by PRRs and initiation of innate immune responses. The balance between proinflammatory responses of cellular PRRs and atheroprotective roles of natural antibodies plays an important role in the development of atherosclerosis. There are likely many more oxidation-specific epitopes that represent such danger-associated molecular patterns and corresponding PRRs that represent respective innate responses. C-PS: Cell wall polysaccharide; OxPL: Oxidized phospholipid; PC: Phosphocholine. Adapted with permission from [26].

Figure 3. The oxidized phospholipid/apoB assay

Microtiter well plates are coated with the murine antibody MB47 and plasma added to bind apoB-100 particles. OxPL on apoB-100 are then detected with biotinylated murine monoclonal antibody E06. LDL: Low-density lipoprotein; Lp(a): Lipoprotein(a); OxPL: Oxidized phospholipid; VLDL: Very low-density lipoprotein.

Figure 4. Levels of oxidized phospholipid/apoB and lipoprotein(a) categorized by racial group

Boxes indicate medians, 25th and 75th percentile, whiskers indicate 10th and 90th percentile. Differences among racial groups are all significant (p < 0.001). BF: Black females; BM: Black males; HF: Hispanic females; HM: Hispanic males; Lp(a): Lipoprotein(a); OxPL: Oxidized phospholipid; RLU: Relative light units; WF: White females; WM: White males. Adapted with permission from [49].

Figure 5. Correlation between oxidized phospholipid/apoB and Lipoprotein(a) in the Dallas Heart Study

(A) Relationship plotted on a geometric scale. (B) Relationship plotted on a logarithmic scale. (C) Relationship in the entire cohort according to apo(a) isoform sizes. Lp(a): Lipoprotein(a); OxPL: Oxidized phospholipid; RLU: Relative light units. Adapted with permission from [49].

Figure 6. Change in oxidized phospholipid/apoB following acute coronary syndromes and uncomplicated percutaneous coronary intervention

(A) Percent change from baseline in OxPL/apoB measured by antibody E06 in patients with ACS. The p-values at the 30-, 120- and 210-day labels represent differences between groups at each time point. Changes in LDL cholesterol are given for comparison. (B) Absolute changes in RLU in oxidized phospholipid/apoB after PCI. p < 0.001 compared with other time points. ACS: Acute coronary syndrome; LDL: Low-density lipoprotein; OxPL: Oxidized phospholipid; PCI: Percutaneous coronary intervention; RLU: Relative light units. Adapted with permission from [27,33].

Figure 7. Odds ratios for obstructive coronary artery disease associated with selected risk factors among patients 60 years of age or younger from the multivariable analysis

Risk factors are shown in descending order of significance. In this analysis, Lp(a) was forced into the model with the OxPL:apoB-100 ratio. CAD: Coronary artery disease; CRP: C-reactive protein; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; Lp(a): Lipoprotein(a); OxPL:apoB-100 ratio: Ratio of oxidized phospholipid content per particle of apoB-100. Adapted with permission from [48].

Figure 8. Multivariate analysis showing the association of oxidized phospholipids/apoB-100 particle tertile groups with the presence and progression of carotid and femoral artery atherosclerosis and with cardiovascular disease

*p < 0.05 for the comparison between the first tertile group (reference category) and the third tertile group. The p-values presented in the figures are the overall p-values for the three tertiles (test for trend). Adapted with permission from [28].

Figure 9. 3D plot of oxidized phospolipid/apoB levels according to lipoprotein(a) mass and apo(a) phenotypes expressed as the number of kringle IV type 2 repeats

The OxPL/apoB levels presented are geometric means (taken as the anti-log of the mean of log-transformed OxPL/apoB values). Lp(a): Lipoprotein(a); OxPL: Oxidized phospolipid. Adapted with permission from [28].

Figure 10. Cumulative hazard curves of incident cardiovascular disease from 1995 to 2005 for tertiles of oxidized phospolipid/apoB in the Bruneck study

OxPL: Oxidized phospholipid. Adapted with permission from [44].

Figure 11. Oxidized phospholipid/apoB ratio within each Framingham Risk Score group

(A) Relationship between tertile groups and cardiovascular disease risk (tertile 1: <0.0379 relative light units (RLU), tertile 2: 0.0379–0.0878 RLU and tertile 3: >0.0878 RLU). (B) Relationship between tertile groups and future coronary artery disease risk (tertile 1: <1150 RLU, tertile 2: 1151–2249 RLU and tertile 3: >2249 RLU). Framingham Risk Score was calculated as low risk (<10% risk of events over 10 years), moderate risk (10–20%) and high risk (>20%). *p< 0.05 and **p< 0.001 for comparison of each tertile of the respective biomarkers with the lowest tertile in the low Framingham Risk Score category of each biomarker. EPIC: European Prospective Investigation of Cancer; Lp(a): Lipoprotein(a); OxPL: Oxidized phospholipid. (A) Adapted with permission from [44]. (B) Adapted with permission from [47].

Figure 12. Odds ratios for fatal and nonfatal coronary artery disease based on tertiles of oxidized phospholipid/apoB and lipoprotein

(a). The tertile cutoffs for OxPL/apoB are tertile 1: <1150 relative light units (RLU), tertile 2: 1151–2249 RLU and tertile 3: >2249 RLU, and for Lp(a) they are tertile 1: <7.25 mg/dl, tertile 2: 7.25–11.69 mg/dl and tertile 3: >11.69 mg/dl. CAD: Coronary artery disease; Lp(a): Lipoprotein(a); OR: Odds ratio; OxPL: Oxidized phospolipid. Adapted with permission from [47].

Figure 13. Relationship of oxidized phospolipid/apoB to lipoprotein-associated phospholipase A₂ and soluble phospholipase A₂ activity

(A) Relationship between OxPL/apoB tertile groups and CVD risk according to tertiles of Lp-PLA₂ activity. (B) ORs for CAD based on tertiles of OxPL/apoB and sPLA₂ activity. The tertile cutoffs for OxPL/apoB are tertile 1: <1150 relative light units (RLU), tertile 2: 1151–2249 RLU and tertile 3: >2249 RLU, and for sPLA₂ activity levels, tertile 1: <4.05 nmol/min/ml, tertile 2: 4.05–4.83 nmol/min/ml and tertile 3: >4.83 nmol/min/ml. CAD: Coronary artery disease; CVD: Cardiovascular disease; EPIC: European Prospective Investigation of Cancer; HR: Hazard ratio; Lp-PLA₂: Lipoprotein-associated phospholipase A₂ activity; OR: Odds ratio; OxPL: Oxidized phospolipid; sPLA₂: Soluble phospholipase A₂. (A) Adapted with permission from [44]. (B) Adapted with permission from [47].

Figure 14. Oxidized phospholipid:apoB ratio and immunohistochemistry in a New Zealand white rabbit study

(A) OxPL:apoB ratio in the New Zealand white rabbit study in the baseline (n = 15), low cholesterol (n = 10) and high cholesterol (n = 5) groups. (B) Immunochemistry of New Zealand white aortas with antibody E06 staining (brown color pattern) for OxPL in the baseline, low cholesterol and high cholesterol diet groups. The arrow represents lack of OxPL at the luminal surface in a representative rabbit with pre-existing atherosclerosis that was subsequently switched to a low-cholesterol diet. OxPL: Oxidized phospholipid. Adapted with permission from [59].