Alexina Orsoni

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.

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia

We measured oxidized phospholipids (OxPL), lipoprotein (a) [Lp(a)], and lipoprotein-associated phospholipase A2 (Lp-PLA2) pre- and postapheresis in 18 patients with familial hypercholesterolemia (FH) and with low(∼10 mg/dl; range 10–11 mg/dl), intermediate (∼50 mg/dl; range 30–61 mg/dl), or high (>100 mg/dl; range 78–128 mg/dl) Lp(a) levels. By using enzymatic and immunoassays, the content of OxPL and Lp-PLA2 mass and activity were quantitated in lipoprotein density fractions plated in microtiter wells, as well as directly on apoB-100, Lp(a), and apoA-I immunocaptured within each fraction (i.e., OxPL/apoB and Lp-PLA2/apoB). In whole fractions, OxPL was primarily detected in the Lp(a)-containing fractions, whereas Lp-PLA2 was primarily detected in the small, dense LDL and light Lp(a) range. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels. Lp-PLA2/apoB and Lp-PLA2/apoA-I levels were highest in the low Lp(a) group but decreased proportionally with increasing Lp(a) levels. Lp-PLA2/apo(a) was lowest in patients with low Lp(a) levels and increased proportionally with increasing Lp(a) levels. Apheresis significantly reduced levels of OxPL and Lp-PLA2 on apoB and Lp(a) (50–75%), particularly in patients with intermediate and high Lp(a) levels. In contrast, apheresis increased Lp-PLA2-specific activity (activity/mass ratio) in buoyant LDL fractions. The impact of apheresis on Lp(a), OxPL, and Lp-PLA2 provides insights into its therapeutic benefits beyond lowering apoB-containing lipoproteins.

LDL-apheresis depletes apoE-HDL and pre-β1-HDL in familial hypercholesterolemia: relevance to atheroprotection.

Subnormal HDL-cholesterol (HDL-C) and apolipoprotein (apo)AI levels are characteristic of familial hypercholesterolemia (FH), reflecting perturbed intravascular metabolism with compositional anomalies in HDL particles, including apoE enrichment. Does LDL-apheresis, which reduces HDL-cholesterol, apoAI, and apoE by adsorption, induce selective changes in HDL subpopulations, with relevance to atheroprotection? Five HDL subpopulations were fractionated from pre- and post-LDL-apheresis plasmas of normotriglyceridemic FH subjects (n = 11) on regular LDL-apheresis (>2 years). Apheresis lowered both plasma apoE (−62%) and apoAI (−16%) levels, with preferential, genotype-independent reduction in apoE. The mass ratio of HDL2:HDL3 was lowered from ∼1:1 to 0.72:1 by apheresis, reflecting selective removal of HDL2 mass (80% of total HDL adsorbed). Pre-LDL-apheresis, HDL2 subpopulations were markedly enriched in apoE, consistent with ∼1 copy of apoE per 4 HDL particles. Large amounts (50-66%) of apoE-HDL were removed by apheresis, preferentially in the HDL2b subfraction (−50%); minor absolute amounts of apoE-HDL were removed from HDL3 subfractions. Furthermore, pre-β1-HDL particle levels were subnormal following removal (−53%) upon apheresis, suggesting that cellular cholesterol efflux may be defective in the immediate postapheresis period. In LDL-receptor (LDL-R) deficiency, LDL-apheresis may enhance flux through the reverse cholesterol transport pathway and equally attenuate potential biglycan-mediated deposition of apoE-HDL in the arterial matrix.

Statin action favors normalization of the plasma lipidome in the atherogenic mixed dyslipidemia of MetS: potential relevance to statin-associated dysglycemia

The impact of statin treatment on the abnormal plasma lipidome of mixed dyslipidemic patients with metabolic syndrome (MetS), a group at increased risk of developing diabetes, was evaluated. Insulin-resistant hypertriglyceridemic hypertensive obese males (n = 12) displaying MetS were treated with pitavastatin (4 mg/day) for 180 days; healthy normolipidemic age-matched nonobese males (n = 12) acted as controls. Statin treatment substantially normalized triglyceride (−41%), remnant cholesterol (−55%), and LDL-cholesterol (−39%), with minor effect on HDL-cholesterol (+4%). Lipidomic analysis, normalized to nonHDL-cholesterol in order to probe statin-induced differences in molecular composition independently of reduction in plasma cholesterol, revealed increment in 132 of 138 lipid species that were subnormal at baseline and significantly shifted toward the control group on statin treatment. Increment in alkyl- and alkenylphospholipids (plasmalogens) was prominent, and consistent with significant statin-induced increase in plasma polyunsaturated fatty acid levels. Comparison of the statin-mediated lipidomic changes in MetS with the abnormal plasma lipidomic profile characteristic of prediabetes and T2D in the Australian Diabetes, Obesity, and Lifestyle Study and San Antonio Family Heart Study cohorts by hypergeometric analysis revealed a significant shift toward the lipid profile of controls, indicative of a marked trend toward a normolipidemic phenotype. Pitavastatin attenuated the abnormal plasma lipidome of MetS patients typical of prediabetes and T2D.

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia.

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (−53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (−15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (−71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (−21%; P < 0.0001) and in the ABCG1-dependent pathway (−15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.

Statin action enriches HDL3 in polyunsaturated phospholipids and plasmalogens and reduces LDL-derived phospholipid hydroperoxides in atherogenic mixed dyslipidemia

Atherogenic mixed dyslipidemia associates with oxidative stress and defective HDL antioxidative function in metabolic syndrome (MetS). The impact of statin treatment on the capacity of HDL to inactivate LDL-derived, redox-active phospholipid hydroperoxides (PCOOHs) in MetS is indeterminate. Insulin-resistant, hypertriglyceridemic, hypertensive, obese males were treated with pitavastatin (4 mg/day) for 180 days, resulting in marked reduction in plasma TGs (−41%) and LDL-cholesterol (−38%), with minor effects on HDL-cholesterol and apoAI. Native plasma LDL (baseline vs. 180 days) was oxidized by aqueous free radicals under mild conditions in vitro either alone or in the presence of the corresponding pre- or poststatin HDL2 or HDL3 at authentic plasma mass ratios. Lipidomic analyses revealed that statin treatment i) reduced the content of oxidizable polyunsaturated phosphatidylcholine (PUPC) species containing DHA and linoleic acid in LDL; ii) preferentially increased the content of PUPC species containing arachidonic acid (AA) in small, dense HDL3; iii) induced significant elevation in the content of phosphatidylcholine and phosphatidylethanolamine (PE) plasmalogens containing AA and DHA in HDL3; and iv) induced formation of HDL3 particles with increased capacity to inactivate PCOOH with formation of redox-inactive phospholipid hydroxide. Statin action attenuated LDL oxidability Concomitantly, the capacity of HDL3 to inactivate redox-active PCOOH was enhanced relative to HDL2, consistent with preferential enrichment of PE plasmalogens and PUPC in HDL3.