Claes Bergmark

Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes.

OBJECTIVES This study was conducted to test the hypothesis that plasma markers of oxidized low-density lipoprotein (OxLDL) reflect acute coronary syndromes (ACS). BACKGROUND Oxidized LDL contributes to the pathogenesis of atherosclerosis, but its role in ACS is not established. METHODS Serial plasma samples were prospectively obtained from patients with an acute myocardial infarction (MI) (n = 8), unstable angina (UA) (n = 15), stable coronary artery disease (CAD) (n = 17), angiographically normal coronary arteries (n = 8), and from healthy subjects (n = 18), at entry into the study, hospital discharge (MI group only), and at 30, 120, and 210 days. Chemiluminescent enzyme-linked immunosorbent assay was used to quantitate plasma levels of: 1) immunoglobulin (Ig)M and IgG OxLDL autoantibody titers (presented as a mean OxLDL autoantibody titer by averaging the results of four distinct epitopes); 2) LDL-autoantibody immune complexes (LDL-IC); and 3) minimally OxLDL measured by antibody E06 (OxLDL-E06), as determined by the content of oxidized phospholipids (OxPL) per apolipoprotein B-100. RESULTS Baseline OxLDL IgG autoantibody levels were higher in the MI group (p < 0.0001). At 30-day follow-up, the mean IgM OxLDL titers increased by 48% (p < 0.001) and 20% (p < 0.001), and IgM LDL-IC increased by 60% (p < 0.01) and 26% (p < 0.01) in the MI and UA groups, respectively. The OxLDL-E06 levels increased by 54% (p < 0.01) in the MI group at hospital discharge and by 36% at 30 days. No significant changes in any OxLDL markers were noted in the other groups. The OxLDL-E06 levels strongly paralleled the acute rise in lipoprotein(a), or Lp(a), in the MI group, suggesting that toxic OxPL are preferentially bound to Lp(a). Oxidized LDL-E06 also correlated extremely well with Lp(a) in the entire cohort of patients (r = 0.91, p < 0.0001). CONCLUSIONS Circulating OxLDL-specific markers strongly reflect the presence of ACS, implying immune awareness to newly exposed oxidation-specific epitopes and possible release of OxLDL in the circulation. The OxLDL-E06 measurements provide novel insights into plaque rupture and the potential atherogenicity of Lp(a).

Lysine-Phosphatidylcholine Adducts in Kringle V Impart Unique Immunological and Potential Pro-inflammatory Properties to Human Apolipoprotein(a)

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

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 ≈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. Conclusion—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.

Induction of Dendritic Cell–Mediated T-Cell Activation by Modified but Not Native Low-Density Lipoprotein in Humans and Inhibition by Annexin A5 Involvement of Heat Shock Proteins

Objective—Atherosclerosis is an inflammatory disease, where activated immunocompetent cells, including dendritic cells (DCs) and T cells are abundant in plaques. Low-density lipoprotein modified either by oxidation (oxLDL) or by human group X-secreted phospholipase A2 (LDLx) and heat shock proteins (HSP), especially HSP60 and 90, have been implicated in atherosclerosis. We previously reported that Annexin A5 inhibits inflammatory effects of phospholipids, decreases vascular inflammation and improves vascular function in apolipoprotein E−/− mice. Here, we focus on the LDLx effects on human DCs and T cells. Approach and Results—Human DCs were differentiated from peripheral blood monocytes, stimulated by oxLDL or LDLx. Naive autologous T cells were cocultured with pretreated DCs. oxLDL and LDLx, in contrast to LDL, induced DC-activation and T-cell proliferation. T cells exposed to LDLx-treated DCs produced interferon-&ggr;, interleukin (IL)-17 but not IL-4 and IL-10. Annexin A5 abrogated LDLx effects on DCs and T cells and increased production of transforming growth factor-&bgr; and IL-10. Furthermore, IL-10 producing T cells suppressed primary T-cell activation via soluble IL-10, transforming growth factor-&bgr;, and cell–cell contact. Lentiviral-mediated shRNA knock-down HSP60 and 90 in DCs attenuated the effect of LDLx on DCs and subsequent T-cell proliferation. Experiments on DC and T cells derived from carotid atherosclerotic plaques gave similar results. Conclusions—Our data show that modified forms of LDL such as LDLx but not native LDL activate human T cells through DCs. HSP60 and 90 contribute to such T-cell activation. Annexin A5 promotes induction of regulatory T cells and is potentially interesting as a therapeutic agent.

Mechanical aortic injury in apoE-deficient mice as a model for development of atherosclerosis: demonstration of leukocyte rolling early after injury.

A mouse model of aortic endothelium regeneration following mechanical injury was studied in wild-type and apoE-deficient (apoE0) animals. The injury induced a topologically nonuniform and complex reparative response. Compared to wild-type animals, apoE0 mice had unaltered ability to regenerate endothelium. Despite the pro-coagulative state of the apoE0 mice, no arterial thromboses were detected. Only deeper arterial injury with damage to the internal elastic membrane was associated with the development of atherosclerotic lesions in apoE0 mice. During the limited observation period of 7 days, superficial arterial injury in apoE0 mice was inconsequential. In addition, for the first time in vivo, rolling of polymorphonuclear leukocytes over the damaged endothelium was documented.