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The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2

Open AccessPublished:October 03, 2013DOI:https://doi.org/10.1194/jlr.M041806
      Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor of cardiovascular disease. Plasma Lp-PLA2 is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA2. We determined the plasma levels of Lp-PLA2-bound apoB (apoB/Lp-PLA2) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA2 concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA2 [apoB/Lp-PLA2(−)] was calculated by subtracting the apoB/Lp-PLA2 from total apoB. The apoB/Lp-PLA2 levels were 3.6-fold higher, while apoB/Lp-PLA2(−) were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA2 and apoB/Lp-PLA2(−) levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA2, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA2(−). Considering that Lp-PLA2 is proatherogenic, the predominance of apoB/Lp-PLA2 particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease.
      Lipoprotein-associated phospholipase A2 (Lp-PLA2) [platelet-activating factor (PAF)- acetylhydrolase] exhibits a Ca2+-independent PLA2 activity and degrades PAF as well as oxidized phospholipids (oxPLs). Such phospholipids are accumulated in the artery wall and may play key roles in vascular inflammation and atherosclerosis. Lp-PLA2 is secreted by macrophages and other inflammatory cells in the arterial wall, and circulates in plasma associated with lipoproteins (
      • Tellis C.C.
      • Tselepis A.D.
      The role of lipoprotein-associated phospholipase A2 in atherosclerosis may depend on its lipoprotein carrier in plasma.
      ). The majority of plasma Lp-PLA2 is bound on apolipoprotein B (apoB)-containing lipoproteins, primarily on low density lipoprotein (LDL) particles. A small proportion of the circulating enzyme is also associated with high density lipoproteins (HDLs). The majority of the LDL-associated Lp-PLA2 is bound to atherogenic small dense LDL (sdLDL) particles (
      • Karabina S.A.
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      PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.
      ). Through hydrolysis of oxPLs, Lp-PLA2 generates pro-inflammatory oxidized nonesterified fatty acids and lysophosphatidylcholine (LPC), which are involved in various stages of atherosclerotic plaque development and may also play an important role in plaque vulnerability. Thus Lp-PLA2 is considered to be a proatherogenic enzyme (
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      Several epidemiologic studies suggest that plasma Lp-PLA2 is an independent predictor of cardiovascular events in primary and secondary prevention (
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      ). A meta-analysis, which included 79,036 participants with or without coronary artery disease (CAD) from 32 prospective studies, showed that Lp-PLA2 activity and mass each had a continuous association with the risk of CAD (
      • Thompson A.
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      ).
      In all epidemiologic studies, the total Lp-PLA2 plasma enzyme has been determined; this mainly represents the LDL-associated Lp-PLA2. Thus plasma Lp-PLA2 is considered as a promising therapeutic target, and two ongoing phase 3 trials on a selective enzyme inhibitor, darapladib, are currently in progress (
      • Tselepis A.F.
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      Therapeutic modulation of lipoprotein-associated phospholipase A2 (Lp-PLA2).
      ). Furthermore, previous studies by our group (
      • Tsimihodimos V.
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      • Chapman M.J.
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      • Tselepis A.D.
      Atorvastatin preferentially reduces LDL-associated platelet-activating factor acetylhydrolase activity in dyslipidemias of type IIA and type IIB.
      ,
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      Differential effect of hypolipidemic drugs on lipoprotein-associated phospholipase A2.
      ) and others (
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      The effect of statin therapy on lipoprotein associated phospholipase A2 levels.
      ,
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      Effects of atorvastatin versus other statins on fasting and postprandial C-reactive protein and lipoprotein-associated phospholipase A2 in patients with coronary heart disease versus control subjects.
      ,
      • Muhlestein J.B.
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      • Lanman R.B.
      • Lavasani F.
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      • Anderson J.L.
      The reduction of inflammatory biomarkers by statin, fibrate, and combination therapy among diabetic patients with mixed dyslipidemia: the DIACOR (Diabetes and Combined Lipid Therapy Regimen) study.
      ) have demonstrated that among existing cardiovascular drugs, statins effectively reduce plasma levels of Lp-PLA2 activity and mass in parallel with the significant reduction of LDL-cholesterol levels, suggesting that the rate of LDL removal from the circulation may represent an important mechanism for the reduction of plasma Lp-PLA2 levels (
      • Tsimihodimos V.
      • Karabina S.A.
      • Tambaki A.P.
      • Bairaktari E.
      • Goudevenos J.A.
      • Chapman M.J.
      • Elisaf M.
      • Tselepis A.D.
      Atorvastatin preferentially reduces LDL-associated platelet-activating factor acetylhydrolase activity in dyslipidemias of type IIA and type IIB.
      ,
      • Saougos V.G.
      • Tambaki A.P.
      • Kalogirou M.
      • Kostapanos M.
      • Gazi I.F.
      • Wolfert R.L.
      • Elisaf M.
      • Tselepis A.D.
      Differential effect of hypolipidemic drugs on lipoprotein-associated phospholipase A2.
      ).
      Importantly, only a proportion of circulating apoB-containing lipoproteins contain Lp-PLA2 (
      • Gazi I.
      • Lourida E.S.
      • Filippatos T.
      • Tsimihodimos V.
      • Elisaf M.
      • Tselepis A.D.
      Lipoprotein-associated phospholipase A2 activity is a marker of small, dense LDL particles in human plasma.
      ). Thus it remains to be established whether these particles differ from those that do not carry Lp-PLA2 in terms of their pathophysiological role in atherosclerosis and their clinical significance, as well as their behavior to pharmacological intervention.
      The aim of the present study was to establish a new method in order to directly determine the plasma concentration of Lp-PLA2-bound apoB (apoB/Lp-PLA2) in normolipidemic healthy volunteers, as well as in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed.

      MATERIALS AND METHODS

      Populational studies

      The study included 53 hypercholesterolemic subjects (30 women and 23 men) and 50 controls (27 women and 23 men). Consecutive patients with primary hypercholesterolemia aged 20 to 70 years attending the Outpatient Lipid and Obesity Clinic of the University Hospital of Ioannina, Greece participated in the present study. Patients were eligible if their LDL-cholesterol levels were above those recommended by the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) based on each patient's risk factors, following a 3 month period of lifestyle changes (

      National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). 2002. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation., 106: 3143–3421.

      ).
      Exclusion criteria were known CAD, symptomatic carotid artery disease, peripheral arterial disease, abdominal aortic aneurysm, diabetes mellitus, renal disease (serum creatinine levels >1.6 mg/dl), hypothyroidism (thyroid stimulating hormone >5 IU/ml), liver disease (alanine aminotranferase and/or aspartate aminotransferase levels >3-fold upper limit of normal in two consecutive measurements), and neoplasia, as well as clinical and laboratory evidence of an inflammatory or infectious conditions within 6 months preceding the study. Patients with hypertension were included in the study if they were on stable medication for at least 3 months and their blood pressure was adequately controlled (no change in their treatment was allowed during the study). Patients taking anti-inflammatory drugs were excluded. Patients currently taking lipid-lowering drugs or having stopped them less than 12 weeks before study entry as well as patients with a history of adverse reactions to statins were excluded. All patients received simvastatin, 40 mg daily, for 3 months. Compliance with treatment was assessed by questionnaire and tablet count. Dietary composition, level of physical activity, smoking habits, and all concurrent medications were maintained unchanged throughout the study period. The control group included 50 age-, sex-, and weight-matched subjects with normal lipid levels [LDL-cholesterol <130 mg/dl, HDL-cholesterol >40 mg/dl, and triglycerides (TGs) <150 mg/dl]. The Ethics Committee of the University Hospital of Ioannina gave approval for the study and all participants gave written consent prior to their enrollment in the study.

      Biochemical parameters

      Venous blood samples were obtained in the morning after 12 h fasting. Concentrations of serum total cholesterol, TGs, and HDL-cholesterol were determined enzymatically on the Olympus AU 600 clinical chemistry analyzer (Olympus Diagnostica, Hamburg, Germany). HDL-cholesterol was determined by a direct assay (Olympus Diagnostica) (
      • Tsimihodimos V.
      • Kakafika A.
      • Tambaki A.P.
      • Bairaktari E.
      • Chapman M.J.
      • Elisaf M.
      • Tselepis A.D.
      Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activity associated with apoB-containing lipoproteins.
      ). LDL-cholesterol was calculated with the Friedewald formula. apoA-I, apoB, and apoE as well as lipoprotein a [Lp(a)] were measured with a Behring BN-100 nephelometer and with reagents (antibodies and calibrators) from Dade Behring Holding GmbH (Liederbach, Germany) (
      • Tsimihodimos V.
      • Kakafika A.
      • Tambaki A.P.
      • Bairaktari E.
      • Chapman M.J.
      • Elisaf M.
      • Tselepis A.D.
      Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activity associated with apoB-containing lipoproteins.
      ). Serum concentrations of high sensitivity C-reactive proteins (hsCRPs) were measured with a high sensitivity immunonephelometric assay (Beckman Instruments, Fullerton, CA). The reference range of this assay is 1.0 to 80 mg/l. The detection limit is 1.0 mg/l. Serum creatinine, liver, and muscle enzymes as well as thyroid function tests were performed by conventional methods, as we have previously described (
      • Tsimihodimos V.
      • Kakafika A.
      • Tambaki A.P.
      • Bairaktari E.
      • Chapman M.J.
      • Elisaf M.
      • Tselepis A.D.
      Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activity associated with apoB-containing lipoproteins.
      ).

      Electrophoretic analysis of apoB-containing lipoprotein subclasses

      Analysis of the apoB-containing lipoprotein subclasses was performed electrophoretically by use of high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL system (Quantimetrix, Redondo Beach, CA), as we have previously described (
      • Gazi I.
      • Lourida E.S.
      • Filippatos T.
      • Tsimihodimos V.
      • Elisaf M.
      • Tselepis A.D.
      Lipoprotein-associated phospholipase A2 activity is a marker of small, dense LDL particles in human plasma.
      ). After electrophoresis, very low density lipoprotein (VLDL) remained in the origin [retention factor (Rf) = 0.0], whereas HDL migrated at the front (Rf = 1.0). In between, several bands were detected: midbands C, B, and A, which correspond mainly to intermediate density lipoprotein (IDL), as well as up to seven LDL bands. The LDL1 and LDL2 bands correspond to large buoyant LDL particles, whereas bands LDL3 to LDL7 correspond to sdLDL particles. We determined the cholesterol mass of each apoB-containing lipoprotein subfraction, the mean LDL particle size (in Å), and the proportion (%) of the cholesterol mass of sdLDL particles over the total LDL cholesterol mass (
      • Gazi I.
      • Lourida E.S.
      • Filippatos T.
      • Tsimihodimos V.
      • Elisaf M.
      • Tselepis A.D.
      Lipoprotein-associated phospholipase A2 activity is a marker of small, dense LDL particles in human plasma.
      ).

      Measurement of plasma Lp-PLA2 activity and mass

      The Lp-PLA2 activity in total plasma was determined by the trichloroacetic acid precipitation procedure using [3H]PAF (100 μmol/l final concentration) as a substrate (
      • Tselepis A.D.
      • Dentan C.
      • Karabina S.A.
      • Chapman M.J.
      • Ninio E.
      PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.
      ). The reaction was performed for 10 min at 37°C. Lp-PLA2 activity was expressed as nanomoles of PAF degraded per minute per milliliter of plasma. The Lp-PLA2 mass in total plasma was determined by a dual monoclonal antibody immunoassay standardized to recombinant Lp-PLA2 (PLAC test kits kindly provided by diaDexus Inc., San Francisco, CA), following the manufacturer's instructions, as we previously described (
      • Hoogeveen R.C.
      • Ballantyne C.M.
      PLAC test for identification of individuals at increased risk for coronary heart disease.
      ).

      Measurement of oxidized LDL

      Plasma levels of oxidized LDL (oxLDL) were measured by a competitive enzyme-linked immunosorbent assay using a specific murine monoclonal antibody 4E6 and following the instructions provided by the manufacturer (Mercodia, Uppsala, Sweden) as we have previously described (
      • Tsouli S.G.
      • Kiortsis D.N.
      • Lourida E.S.
      • Xydis V.
      • Tsironis L.D.
      • Argyropoulou M.I.
      • Elisaf M.
      • Tselepis A.D.
      Autoantibody titers against OxLDL are correlated with Achilles tendon thickness in patients with familial hypercholesterolemia.
      ). Intra- and interassay coefficients of variation of the assay were 6.0% and 7.0%, respectively.

      Determination of apoB/Lp-PLA2

      To quantify the apoB/Lp-PLA2 plasma levels, we established an enzyme-linked immunosorbent assay (ELISA) as follows. Microtiter 96-well plates (Costar, Corning Inc., NY) were coated overnight at 4°C with 100 μl/well of the anti-Lp-PLA2 monoclonal antibody 2C10 (kindly provided by diaDexus Inc.) in 0.2 mol/l sodium phosphate buffer, pH 6.5 (10 μg protein/ml). Wells were then washed four times with a 50 mmol/l Tris-HCl buffer saline solution, pH 7.4 (TBS) containing 0.1% Tween-20 to remove any excess antibody, and the noncoated surface was then blocked by an overnight incubation at room temperature with 300 μl/well of a blocking solution containing 1% free fatty acid-bovine serum albumin (FFA-BSA) and 2.5% sucrose solution in 10 mmol/l potassium phosphate buffer, pH 7.4. Subsequently, the blocking solution was completely removed, and the coated plates were air-dried at room temperature, sealed with desiccant, and stored at 4°C. The apoB/Lp-PLA2 levels were determined using 40 μl plasma samples that were added to each well, and the volume was completed to 200 μl with assay buffer that consisted of 0.2% FFA-BSA, 0.2% mouse serum, 0.2% calf serum, and 0.1% Proclin-150 in TBS (pH 7.4). A calibration curve was simultaneously prepared using LDL at a range from 1 to 60 μg apoB. Wells containing either plasma samples or LDL calibrators were incubated for 90 min at room temperature on a plate shaker at 600 rpm and then washed four times with TBS. Two hundred microliters of an anti-apoB horseradish peroxidase (HRP)-conjugated polyclonal antibody (1 mg/ml; Acris Antibodies GmbH, Herford, Germany), diluted 1:10,000 v/v with assay buffer (final concentration 0.1 μg protein/ml), were added to each well and incubated for 90 min at room temperature on a plate shaker at 600 rpm. Subsequently, the plate was washed four times with a 0.025% Tween-20 solution in TBS and then 100 μl of a HRP substrate (TMB, 3,3′, 5,5″-tetramethylbenzidine; Cell Signaling Inc., Danvers, MA) were added to each well. The plate was incubated at room temperature for 20 min in the dark. The reaction was stopped with 100 μl/well 1N HCl. The optical density of plasma samples and LDL calibrators was measured versus blank (100 μl of a HRP substrate plus 100 μl 1N HCl) at 450 nm using a microwell plate reader. Negative controls were also prepared by following the above procedure in wells which were not covered with the anti-Lp-PLA2 monoclonal antibody 2C10.

      Preparation of LDL calibrators.

      The LDL used for the calibration curve was isolated from pooled fresh plasma from healthy normolipidemic volunteers by sequential ultracentrifugation at d = 1.019–1.063 g/ml (
      • Liapikos T.A.
      • Antonopoulou S.
      • Karabina S.P.
      • Tsoukatos D.C.
      • Demopoulos C.A.
      • Tselepis A.D.
      Platelet-activating factor formation during oxidative modification of low-density lipoprotein when PAF-acetylhydrolase has been inactivated.
      ). LDL protein was determined by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL) (
      • Smith P.K.
      • Krohn R.I.
      • Hermanson G.T.
      • Mallia A.K.
      • Gartner F.H.
      • Provenzano M.D.
      • Fujimoto E.K.
      • Goeke N.M.
      • Olson B.J.
      • Klenk D.C.
      Measurement of protein using bicinchoninic acid.
      ). The Lp-PLA2 mass content of LDL was determined by the PLAC® test (diaDexus Inc.) using 10 μg of LDL protein, whereas the LDL apoB content was determined with a Behring Holding GmbH BN-100 nephelometer (Liederbach Inc., Germany) (
      • Steinmetz J.
      • Tarallo P.
      • Fournier B.
      • Caces E.
      • Siest G.
      Reference limits of apolipoprotein A-I and apolipoprotein B using an IFCC standardized immunonephelometric method.
      ). In some experiments oxLDL instead of native LDL was used as a calibrator. LDL was oxidized in the presence of copper sulfate (5 μM final concentration). The kinetics of oxidation was determined by continuously monitoring the increase in absorbance at 234 nm for 6 h, as we have previously described (
      • Karabina S.A.
      • Liapikos T.A.
      • Grekas G.
      • Goudevenos J.
      • Tselepis A.D.
      Distribution of PAF-acetylhydrolase activity in human plasma low-density lipoprotein subfractions.
      ,
      • Tselepis A.
      • Doulias P.
      • Lourida E.
      • Glantzounis G.
      • Tsimoyiannis E.
      • Galaris D.
      Trimetazidine protects low-density lipoproteins from oxidation and cultured cells exposed to H(2)O(2) from DNA damage.
      ). The purity and the oxidative modification of the LDL preparations were evaluated by agarose gel electrophoresis (Hydragel Lipo and Lp(a) kit, Sebia Inc., France) (
      • Tselepis A.
      • Doulias P.
      • Lourida E.
      • Glantzounis G.
      • Tsimoyiannis E.
      • Galaris D.
      Trimetazidine protects low-density lipoproteins from oxidation and cultured cells exposed to H(2)O(2) from DNA damage.
      ). The LDL calibrators as well as the oxLDL preparations were stored in the dark in sealed tubes overlaid with N2 at 4°C and used within 2 weeks. The total protein content of various LDL preparations ranged from 2.5 to 3.5 mg/ml, the Lp-PLA2 mass ranged from 5 to 10 ng/mg protein, and the apoB levels ranged from 2 to 3 mg/ml. In selected experiments HDL (d = 1.063–1.210 g/ml, prepared by sequential ultracentrifugation as we have previously described (
      • Kakafika A.I.
      • Xenofontos S.
      • Tsimihodimos V.
      • Tambaki A.P.
      • Lourida E.S.
      • Kalaitzidis R.
      • Cariolou M.A.
      • Elisaf M.
      • Tselepis A.D.
      The PON1 M55L gene polymorphism is associated with reduced HDL-associated PAF-AH activity.
      ) was used instead of LDL, at a range from 1 to 60 μg apoA-I.

      Statistical analysis

      Data are presented as the mean ± standard deviation (SD) and median (range) for parametric and nonparametric data, respectively. The differences of study parameters between controls and hypercholesterolemic participants, as well as baseline and posttreatment values for patients, were evaluated by paired samples t-tests (or Wilcoxon's rank test for non-Gaussian variables). Significance was defined at P < 0.05 (two-tailed). Analyses were performed using the Statistical Package for the Social Sciences (SPSS) 16.0 (SPSS Inc., Chicago, IL).

      RESULTS

      Clinical and biochemical characteristics of the study population

      Fifty-three hypercholesterolemic subjects (30 women and 23 men, aged 57 ± 13 years) and 50 controls (27 women and 23 men, aged 54 ± 11 years) participated in the study. The clinical and biochemical characteristics of the study population are shown in Table 1. Hypercholesterolemic patients exhibited significantly higher body mass index values as well higher serum levels of total cholesterol, LDL-cholesterol, oxLDLs, and hsCRPs compared with controls. Hypercholesterolemic patients also exhibited higher levels of buoyant LDL-cholesterol and sdLDL-cholesterol compared with controls, whereas no difference in the TGs, Lp(a), sdLDL proportion, and mean LDL size was observed between the two study groups (Table 1). apoB as well as Lp-PLA2 activity and mass levels were also significantly higher in hypercholesterolemic patients compared with controls, whereas no difference in the ratio of Lp-PLA2 mass/apoB was observed between hypercholesterolemic patients and controls (Table 1).
      TABLE 1Clinical and biochemical characteristics of control subjects and hypercholesterolemic patients at baseline and 3 months posttreatment with simvastatin
      Simvastatin (n = 53)
      ParameterControls (n = 50)BaselinePosttreatment
      Age (years)54 ± 1157 ± 13
      Sex (male/female)23/2723/30
      BMI (kg/m2)25 ± 329 ± 5*28 ± 3*
      Current smoking (n)
      Current smoking was defined as smoking at least one cigarette daily.
      1518
      Total cholesterol (mg/dl)165 ± 30257 ± 40*175 ± 30**
      TGs (mg/dl)101 ± 26112 ± 5797 ± 32
      HDL-cholesterol (mg/dl)58 ± 757 ± 1358 ± 11
      LDL-cholesterol (mg/dl)107 ± 26176 ± 34*97 ± 26**
      Buoyant LDL-cholesterol (mg/dl)68 ± 1688 ± 20*51 ± 17**
      sdLDL-cholesterol (mg/dl)5 (0–18)9 (4–22)*5 (0–16)**
      sdLDL proportion (%)4.9 ± 1.95.1 ± 2.35.2 ± 2.0
      Mean LDL size, nm26.9 ± 0.226.6 ± 0.326.7 ± 0.4
      apoB (mg/dl)76 ± 17113 ± 24*72 ± 19**
      apoA-I (mg/dl)151 ± 19154 ± 26158 ± 24
      apoE (mg/dl)40 ± 844 ± 1041 ± 8
      oxLDL (U/l)45 ± 1289 ± 18*55 ± 20**
      Lp(a) (mg/dl)6.8 (2.3–19.1)7.5 (2.3–21.3)7.3 (2.4–25.4)
      hsCRP (mg/l)2.2 (1.0–6.4)3.0 (1.2–12.9)*2.5 (1.0–9.7) **
      Lp-PLA2 activity (nmol/ml/min)48 ± 964 ± 16*44 ± 16**
      Lp-PLA2 mass (ng/ml)261 ± 41397 ± 77**251 ± 71**
      Lp-PLA2 mass/apoB ratio (ng/mg)344 ± 26351 ± 35349 ± 39
      Values are expressed as mean ± SD except for Lp(a) and hsCRP which are expressed as median (range). *P < 0.005 compared with controls; **P < 0.001 compared with baseline values. BMI, body mass index.
      a Current smoking was defined as smoking at least one cigarette daily.

      Effect of simvastatin therapy on lipid parameters and Lp-PLA2

      As expected, simvastatin therapy significantly reduced serum levels of total cholesterol, LDL-cholesterol, and oxLDLs (Table 1). Furthermore, simvastatin significantly reduced buoyant LDL-cholesterol and sdLDL-cholesterol levels, but it did not affect sdLDL proportion and mean LDL size (Table 1). Finally, simvastatin significantly decreased hsCRP and apoB levels as well as Lp-PLA2 activity and mass; however, it did not affect the ratio of Lp-PLA2 mass/apoB (Table 1).

      Calibration curve for apoB/Lp-PLA2 determination

      For the determination of plasma apoB/Lp-PLA2 levels, we established an ELISA method in which microtiter 96-well plates were coated with the anti-Lp-PLA2 monoclonal antibody 2C10 to capture the plasma Lp-PLA2-containing lipoprotein particles. An anti-apoB HRP-conjugated polyclonal antibody was subsequently used to specifically quantify the plasma apoB-containing lipoproteins that were captured by the anti-Lp-PLA2 monoclonal antibody. Different concentrations of LDL, isolated by sequential ultracentrifugation from pooled fresh plasma of healthy normolipidemic volunteers, were used as calibrators. A standard point-to-point calibration curve was constructed by plotting the absorbance obtained for each LDL calibrator on the vertical axis versus the mass of LDL calibrator expressed as apoB mass in micrograms on the horizontal axis (Fig. 1). No absorbance against blank was observed in wells used as negative controls or in wells where HDL was placed instead of LDL (data not shown). To evaluate whether LDL oxidation influences the apoB/Lp-PLA2 assay, we used oxLDL instead of native LDL as a calibrator. As is shown in Fig. 1, oxidation of LDL does not affect the calibration curve for apoB/Lp-PLA2 determination. The minimum detection limit of this method is 0.9 μg/ml, whereas the intra- and inter-assay coefficients of variation are 7.6 and 9.1%, respectively.
      Figure thumbnail gr1
      Fig. 1A standard calibration curve for the determination of apoB/Lp-PLA2 levels. This curve was constructed by plotting the absorbance obtained for each calibrator (LDL or oxLDL) on the y axis versus the amount of LDL or oxLDL, respectively, expressed in micrograms of apoB.

      apoB/Lp-PLA2 levels in the study population and effect of simvastatin therapy

      Using the standard calibration curve, we determined the concentration of apoB/Lp-PLA2 in our plasma samples (expressed as apoB levels in milligrams per deciliter). These apoB levels correspond to the concentration of the Lp-PLA2-bound apoB. The concentration of the apoB lipoproteins that do not carry Lp-PLA2 [apoB/Lp-PLA2(−)] was calculated by subtracting the apoB/Lp-PLA2 from total apoB levels. The present study shows for the first time that apoB/Lp-PLA2 are significantly higher (3.6-fold) in hypercholesterolemic patients compared with controls (Fig. 2A). apoB/Lp-PLA2 in hypercholesterolemic patients represents the 22.1% of total apoB levels while in normolipidemic controls it represents the 9.2% of total apoB. The apoB/Lp-PLA2(−) are modestly, albeit significantly, higher (1.3-fold) compared with controls (Fig. 2A), and this increase is similar to that of total apoB (1.5-fold increase). Furthermore, the ratio of apoB/Lp-PLA2/apoB in hypercholesterolemic patients is significantly higher compared with normolipidemic controls (Fig. 2B), whereas the ratio of apoB/Lp-PLA2(−)/apoB was modestly, albeit significantly, lower in hypercholesterolemic patients than in controls, primarily due to the relatively higher elevation of apoB compared with apoB/Lp-PLA2(−) in hypercholesterolemic patients (Fig. 2B). These results suggest that among apoB-containing lipoproteins, the particles that are mostly elevated in hypercholesterolemic patients at baseline are apoB/Lp-PLA2 particles.
      Figure thumbnail gr2
      Fig. 2A: Bar graph showing the total apoB, apoB/Lp-PLA2 (−) and apoB/Lp-PLA2 levels in normolipidemic controls as well as in hypercholesterolemic patients at baseline, as well as after 3 months of treatment with 40 mg/day of simvastatin. Data are mean ± SD. *P < 0.005 and **P < 0.001 compared with the control group. # P < 0.001 and $ P < 0.005 compared with baseline values. B: Bar graph showing the ratios of apoB/Lp-PLA2/apoB, and apoB/Lp-PLA2(−)/apoB in normolipidemic controls as well as in hypercholesterolemic patients at baseline and after 3 months of treatment with 40 mg/day of simvastatin. Data are mean ± SD. *P < 0.001 and **P < 0.05 compared with the control group; # P < 0.001 compared with baseline values.
      After 3 months of treatment with simvastatin, the plasma apoB/Lp-PLA2 levels were significantly reduced by 52% compared with baseline, whereas the apoB/Lp-PLA2(−) levels were reduced by 33% (Fig. 2A). It should be mentioned that simvastatin reduced the total apoB levels by 36% (Table 1). Consequently, the apoB/Lp-PLA2/apoB ratio was significantly reduced by simvastatin, whereas the ratio of apoB/Lp-PLA2(−)/apoB was not significantly altered (Fig. 2B).

      Correlations

      apoB/Lp-PLA2 and apoB/Lp-PLA2(−) levels in controls as well as in hypercholesterolemic patients at baseline were positively correlated with total cholesterol, TGs, LDL-cholesterol, apoB, Lp(a), sdLDL-cholesterol, buoyant LDL-cholesterol, oxLDL levels, and sdLDL proportion (Table 2). apoB/Lp-PLA2, but not apoB/Lp-PLA2(−), levels were positively correlated with hsCRP levels as well as Lp-PLA2 mass and activity (Table 2). The changes in plasma apoB/Lp-PLA2 levels in response to simvastatin therapy in hypercholesterolemic patients were positively correlated with the changes in total cholesterol, LDL-cholesterol, apoB, sdlDL-cholesterol, buoyant LDL-cholesterol, and oxLDL, as well as with hsCRP, Lp-PLA2 mass, and activity (Table 3).
      TABLE 2Correlation between plasma apoB/Lp-PLA2 and apoB/Lp-PLA2(−) levels and lipid inflammatory parameters in the total population
      apoB/Lp-PLA2apoB/Lp-PLA2(−)
      ParametersrPrP
      Total cholesterol0.692<0.0010.801<0.001
      TGs0.389<0.010.286<0.03
      HDL-cholesterol−0.178NS−0.003NS
      LDL-cholesterol0.732<0.0010.830<0.001
      apoA-1−0.104NS0.097NS
      apoB0.679<0.0010.897<0.001
      apoE0.190NS0.300<0.03
      Lp(a)0.262<0.010.241<0.01
      sdLDL-cholesterol0.248<0.030.278<0.02
      sdLDL proportion0.464<0.0010.473<0.001
      Buoyant LDL-cholesterol0.498<0.0010.480<0.001
      oxLDL0.452<0.0010.571<0.001
      hsCRP0.264<0.020.081NS
      LpPLA2 mass0.786<0.0010.081NS
      LpPLA2 activity0.703<0.0010.066NS
      TABLE 3Correlation between percent changes of plasma apoB/Lp-PLA2 levels and lipid as well as inflammatory parameters after simvastatin treatment in hypercholesterolemic patients
      apoB/Lp-PLA2 (% Change)
      ParametersrP
      Total cholesterol0.314<0.03
      TGs0.058NS
      LDL-cholesterol0.490<0.001
      apoB0.377<0.005
      Lp(a)0.049NS
      sdLDL-cholesterol0.247<0.03
      Buoyant LDL-cholesterol0.463<0.004
      oxLDL0.513<0.003
      hsCRP0.321<0.01
      Lp-PLA2 mass0.554<0.001
      Lp-PLA2 activity0.496<0.001

      DISCUSSION

      In the present study, we propose an ELISA method to quantify the Lp-PLA2-bound apoB in human plasma. This method is specific for apoB-containing lipoproteins, primarily LDLs, because apoA-I-containing HDL particles do not have any reactivity. According to the present results, apoB/Lp-PLA2 represents 9.2% of total apoB levels in controls. In a previous study (
      • Gazi I.
      • Lourida E.S.
      • Filippatos T.
      • Tsimihodimos V.
      • Elisaf M.
      • Tselepis A.D.
      Lipoprotein-associated phospholipase A2 activity is a marker of small, dense LDL particles in human plasma.
      ), we had reported that only a small proportion of circulating apoB-containing lipoproteins carry Lp-PLA2. According to our calculations, one molecule of Lp-PLA2 corresponds to approximately 100 sdLDL (LDL5) and to 4,000 buoyant LDL (LDL2 and LDL3) particles (
      • Gazi I.
      • Lourida E.S.
      • Filippatos T.
      • Tsimihodimos V.
      • Elisaf M.
      • Tselepis A.D.
      Lipoprotein-associated phospholipase A2 activity is a marker of small, dense LDL particles in human plasma.
      ). In this regard, we should point out that only one molecule of Lp-PLA2 can bind (mainly through residues Tyr-205, Trp-115, and Leu-116) to one molecule of apoB (at residues 4119 to 4279 of the carboxyl terminus) and thus to one LDL particle (
      • Stafforini D.M.
      • Tjoelker L.W.
      • McCormick S.P.
      • Vaitkus D.
      • McIntyre T.M.
      • Gray P.W.
      • Young S.G.
      • Prescott S.M.
      Molecular basis of the interaction between plasma platelet-activating factor acetylhydrolase and low density lipoprotein.
      ). The underestimation of the amount of Lp-PLA2 associated with apoB-containing lipoproteins in our previous study, compared with the present study, is most likely attributed to the fact that in the previous study we used density gradient ultracentrifugation to subfractionate plasma lipoproteins. During ultracentrifugation, a proportion of enzyme dissociates from apoB-containing lipoprotein particles and is recovered in the very dense portion of the gradient with proteins (
      • McCall M.R.
      • La Belle M.
      • Forte T.M.
      • Krauss R.M.
      • Takanami Y.
      • Tribble D.L.
      Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility.
      ).
      The present study further shows that LDL oxidation does not affect the assay of apoB/Lp-PLA2 because the standard plots are similar when we use either native LDLs or oxLDLs as calibrators. This is in line with the significant positive correlations obtained between apoB/Lp-PLA2 and LDL-cholesterol or oxLDL levels. We and others have previously demonstrated that Lp-PLA2 is also associated with Lp(a) in human plasma (
      • Karabina S.A.
      • Elisaf M.C.
      • Goudevenos J.
      • Siamopoulos K.C.
      • Sideris D.
      • Tselepis A.D.
      PAF-acetylhydrolase activity of Lp(a) before and during Cu(2+)-induced oxidative modification in vitro.
      ,
      • Tsironis L.D.
      • Katsouras C.S.
      • Lourida E.S.
      • Mitsios J.V.
      • Goudevenos J.
      • Elisaf M.
      • Tselepis A.D.
      Reduced PAF-acetylhydrolase activity associated with Lp(a) in patients with coronary artery disease.
      ,
      • Blencowe C.
      • Hermetter A.
      • Kostner G.M.
      • Deigner H.P.
      Enhanced association of platelet-activating factor acetylhydrolase with lipoprotein (a) in comparison with low density lipoprotein.
      ). Lp(a) contains 1.5- to 2-fold higher enzyme mass (
      • Karabina S.A.
      • Elisaf M.C.
      • Goudevenos J.
      • Siamopoulos K.C.
      • Sideris D.
      • Tselepis A.D.
      PAF-acetylhydrolase activity of Lp(a) before and during Cu(2+)-induced oxidative modification in vitro.
      ,
      • Tsironis L.D.
      • Katsouras C.S.
      • Lourida E.S.
      • Mitsios J.V.
      • Goudevenos J.
      • Elisaf M.
      • Tselepis A.D.
      Reduced PAF-acetylhydrolase activity associated with Lp(a) in patients with coronary artery disease.
      ) and several-fold greater Lp-PLA2 activity compared with LDL when assayed at equimolar protein concentrations (
      • Blencowe C.
      • Hermetter A.
      • Kostner G.M.
      • Deigner H.P.
      Enhanced association of platelet-activating factor acetylhydrolase with lipoprotein (a) in comparison with low density lipoprotein.
      ). In the present study apoB/Lp-PLA2 is positively correlated with Lp(a), a finding consistent with the suggestion that the present method also determines Lp(a) particles containing Lp-PLA2. However, this correlation is relatively weak, possibly due to the low plasma Lp(a) levels in our population. Indeed, we have previously demonstrated that the distribution of Lp-PLA2 between LDLs and HDLs can be influenced by the presence of Lp(a) only when plasma levels of this lipoprotein exceed 30 mg/dl (
      • Karabina S.A.
      • Elisaf M.C.
      • Goudevenos J.
      • Siamopoulos K.C.
      • Sideris D.
      • Tselepis A.D.
      PAF-acetylhydrolase activity of Lp(a) before and during Cu(2+)-induced oxidative modification in vitro.
      ). Thus detectable amounts of Lp(a)-associated Lp-PLA2 in plasma can be obtained with at least three times higher Lp(a) concentration than that observed in the present study.
      Lp-PLA2 is also associated with the TG-rich VLDLs and IDLs representing only 3.3 ± 1.6% in human plasma (
      • Tselepis A.D.
      • Dentan C.
      • Karabina S.A.
      • Chapman M.J.
      • Ninio E.
      PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.
      ). The significant correlation observed between apoB/Lp-PLA2 and TG levels is consistent with the suggestion that the present method also determines VLDL+IDL particles containing Lp-PLA2. By contrast, apoB/Lp-PLA2 was not correlated with either apoA-I or HDL-cholesterol levels, which accords with the lack of any reactivity of apoA-I-containing HDL particles in the calibration curve. Overall, the present method specifically determines the apoB-containing lipoproteins carrying Lp-PLA2 in human plasma, primarily LDLs. Because our population exhibits low Lp(a) and TG levels, further studies are required in a population with high Lp(a) and TG levels to further support the suggestion that this method also determines Lp(a)-associated Lp-PLA2 and VLDL+IDL-associated Lp-PLA2.
      An important observation of the present study is that the increase in the apoB/Lp-PLA2 in hypercholesterolemic patients at baseline is relatively higher than that of apoB/Lp-PLA2(−) (3.6-fold vs. 1.3-fold, respectively, compared with controls). Thus we may suggest that the defective metabolism of apoB-containing lipoproteins observed in primary hypercholesterolemia mainly reflects the apoB/Lp-PLA2 particles and could be primarily attributed to the elevation of LDL and oxLDL particles, because neither TG (VLDL+IDL) nor Lp(a) levels are increased in hypercholesterolemic patients. This is further supported by the positive correlation between the reduction of apoB/Lp-PLA2 and LDL-cholesterol or oxLDL levels induced by simvastatin. A feature characteristic of patients with primary hypercholesterolemia is the reduction in the rate of LDL removal from the circulation (
      • Brown M.S.
      • Goldstein J.L.
      Receptor-mediated control of cholesterol metabolism.
      ). The present study suggests that this metabolic LDL abnormality, which leads to the increase in plasma LDL-cholesterol levels, predominantly reflects the LDL particles carrying Lp-PLA2. Our study does not provide the biochemical basis for this phenomenon. However, we may suggest that Lp-PLA2 could be involved in the LDL receptor-dependent and -independent pathways of LDL catabolism. In line with this hypothesis are the results of a recent study showing that cholesteryl ester transfer between HDLs and apoB-containing lipoproteins in plasma may be positively influenced by Lp-PLA2 (
      • Constantinides A.
      • Kerstens M.N.
      • Dikkeschei B.D.
      • van Pelt L.J.
      • Tellis C.C.
      • Tselepis A.D.
      • Dullaart R.P.
      Plasma Lp-PLA(2) mass and apoB-lipoproteins that carry Lp-PLA(2) decrease after sodium.
      ), suggesting a new proatherogenic function of this enzyme. The above hypothesis needs to be further elucidated. apoB/Lp-PLA2 is positively correlated with sdLDL-cholesterol levels, and previous studies have shown that sdLDL particles undergo decreased recognition by the LDL receptor, thus resulting in lower clearance rates from the circulation and in their increased plasma half-life (
      • Nigon F.
      • Lesnik P.
      • Rouis M.
      • Chapman M.J.
      Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor.
      ,
      • Galeano N.F.
      • Milne R.
      • Marcel Y.L.
      • Walsh M.T.
      • Levy E.
      • Ngu'yen T.D.
      • Gleeson A.
      • Arad Y.
      • Witte L.
      • Al-Haideri M.
      • et al.
      Apoprotein B structure and receptor recognition of triglyceride-rich low density lipoprotein (LDL) is modified in small LDL but not in triglyceride-rich LDL of normal size.
      ,
      • Galeano N.F.
      • Al-Haideri M.
      • Keyserman F.
      • Rumsey S.C.
      • Deckelbaum R.J.
      Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity.
      ,
      • Toyota Y.
      • Yamamura T.
      • Miyake Y.
      • Yamamoto A.
      Low density lipoprotein (LDL) binding affinity for the LDL receptor in hyperlipoproteinemia.
      ). Because sdLDL particles are enriched in Lp-PLA2 (
      • Karabina S.A.
      • Liapikos T.A.
      • Grekas G.
      • Goudevenos J.
      • Tselepis A.D.
      Distribution of PAF-acetylhydrolase activity in human plasma low-density lipoprotein subfractions.
      ,
      • Tselepis A.D.
      • Dentan C.
      • Karabina S.A.
      • Chapman M.J.
      • Ninio E.
      PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.
      ), we suggest that this enzyme may play a role in the decreased clearance rates of sdLDLs.
      apoB/Lp-PLA2 at baseline is positively correlated with plasma Lp-PLA2 activity and mass which are significantly reduced by simvastatin. This finding is in accordance with previously published results by our group (
      • Tsimihodimos V.
      • Karabina S.A.
      • Tambaki A.P.
      • Bairaktari E.
      • Goudevenos J.A.
      • Chapman M.J.
      • Elisaf M.
      • Tselepis A.D.
      Atorvastatin preferentially reduces LDL-associated platelet-activating factor acetylhydrolase activity in dyslipidemias of type IIA and type IIB.
      ,
      • Saougos V.G.
      • Tambaki A.P.
      • Kalogirou M.
      • Kostapanos M.
      • Gazi I.F.
      • Wolfert R.L.
      • Elisaf M.
      • Tselepis A.D.
      Differential effect of hypolipidemic drugs on lipoprotein-associated phospholipase A2.
      ) and others (
      • Albert M.A.
      • Glynn R.J.
      • Wolfert R.L.
      • Ridker P.M.
      The effect of statin therapy on lipoprotein associated phospholipase A2 levels.
      ,
      • Schaefer E.J.
      • McNamara J.R.
      • Asztalos B.F.
      • Tayler T.
      • Daly J.A.
      • Gleason J.L.
      • Seman L.J.
      • Ferrari A.
      • Rubenstein J.J.
      Effects of atorvastatin versus other statins on fasting and postprandial C-reactive protein and lipoprotein-associated phospholipase A2 in patients with coronary heart disease versus control subjects.
      ,
      • Muhlestein J.B.
      • May H.T.
      • Jensen J.R.
      • Horne B.D.
      • Lanman R.B.
      • Lavasani F.
      • Wolfert R.L.
      • Pearson R.R.
      • Yannicelli H.D.
      • Anderson J.L.
      The reduction of inflammatory biomarkers by statin, fibrate, and combination therapy among diabetic patients with mixed dyslipidemia: the DIACOR (Diabetes and Combined Lipid Therapy Regimen) study.
      ), demonstrating that statins effectively reduce plasma levels of Lp-PLA2. The present study further shows that simvastatin significantly reduces apoB/Lp-PLA2 levels, this reduction being positively correlated with that of plasma Lp-PLA2 activity and mass. The above correlations prompted us to suggest that the increase in apoB/Lp-PLA2 observed in hypercholesterolemic patients may also be attributed to the increased plasma Lp-PLA2 levels.
      Circulating Lp-PLA2 derives primarily from cells of the hematopoietic lineage, mainly monocyte-derived macrophages (
      • Asano K.
      • Okamoto S.
      • Fukunaga K.
      • Shiomi T.
      • Mori T.
      • Iwata M.
      • Ikeda Y.
      • Yamaguchi K.
      Cellular source(s) of platelet-activating-factor acetylhydrolase activity in plasma.
      ,
      • Tselepis A.D.
      • Karabina S.A.
      • Stengel D.
      • Piedagnel R.
      • Chapman M.J.
      • Ninio E.
      N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL.
      ), i.e., cells that do not produce apoB-containing lipoproteins. Thus, the release of Lp-PLA2 occurs independently of lipoprotein secretion; the enzyme subsequently associates with these particles (
      • Tellis C.C.
      • Tselepis A.D.
      The role of lipoprotein-associated phospholipase A2 in atherosclerosis may depend on its lipoprotein carrier in plasma.
      ). Lp-PLA2 is actively produced and secreted by monocyte-derived macrophages in response to inflammatory cytokines and microbial lipopolysaccharides (
      • Macphee C.H.
      • Nelson J.J.
      • Zalewski A.
      Lipoprotein-associated phospholipase A2 as a target of therapy.
      ,
      • Wu X.
      • Zimmerman G.A.
      • Prescott S.M.
      • Stafforini D.M.
      The p38 MAPK pathway mediates transcriptional activation of the plasma platelet-activating factor acetylhydrolase gene in macrophages stimulated with lipopolysaccharide.
      ,
      • Memon R.A.
      • Fuller J.
      • Moser A.H.
      • Feingold K.R.
      • Grunfeld C.
      In vivo regulation of plasma platelet-activating factor acetylhydrolase during the acute phase response.
      ,
      • Svetlov S.I.
      • Sturm E.
      • Olson M.S.
      • Crawford J.M.
      Hepatic regulation of platelet-activating factor acetylhydrolase and lecithin:cholesterol acyltransferase biliary and plasma output in rats exposed to bacterial lipopolysaccharide.
      ,
      • Howard K.M.
      • Olson M.S.
      The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats.
      ). Hypercholesterolemic patients exhibit a low grade inflammation, as is indicated by the higher hsCRP levels compared with controls, a finding that is in accordance with our previously published results (
      • Moutzouri E.
      • Tellis C.C.
      • Rousouli K.
      • Liberopoulos E.N.
      • Milionis H.J.
      • Elisaf M.S.
      • Tselepis A.D.
      Effect of simvastatin or its combination with ezetimibe on Toll-like receptor expression and lipopolysaccharide - induced cytokine production in monocytes of hypercholesterolemic patients.
      ). hsCRP levels are positively correlated with apoB/Lp-PLA2 levels while the percent reduction of hsCRP induced by simvastatin is positively correlated with that of apoB/Lp-PLA2. In this regard it has been demonstrated that simvastatin effectively reduces Lp-PLA2 expression and secreted activity in macrophages primarily during inflammatory stimulation with lipopolysaccharides, through inhibition of the mevalonate-geranylgeranyl pyrophosphate-RhoA-p38 mitogen-activated protein kinase pathway (
      • Song J.X.
      • Ren J.Y.
      • Chen H.
      Simvastatin reduces lipoprotein-associated phospholipase A2 in lipopolysaccharide-stimulated human monocyte-derived macrophages through inhibition of the mevalonate-geranylgeranyl pyrophosphate-RhoA-p38 mitogen-activated protein kinase pathway.
      ). It should be noted that this reduction by statins is not observed in the absence of inflammatory stimuli, a finding that accords with previously published data by our group and others (
      • Tsimihodimos V.
      • Karabina S.A.
      • Tambaki A.P.
      • Bairaktari E.
      • Goudevenos J.A.
      • Chapman M.J.
      • Elisaf M.
      • Tselepis A.D.
      Atorvastatin preferentially reduces LDL-associated platelet-activating factor acetylhydrolase activity in dyslipidemias of type IIA and type IIB.
      ,
      • Zhang B.
      • Fan P.
      • Shimoji E.
      • Itabe H.
      • Miura S.
      • Uehara Y.
      • Matsunaga A.
      • Saku K.
      Modulating effects of cholesterol feeding and simvastatin treatment on platelet-activating factor acetylhydrolase activity and lysophosphatidylcholine concentration.
      ). Thus it is possible that the low grade inflammation occurring in our hypercholesterolemic patients may lead to increased secretion of Lp-PLA2 and subsequently to increased levels of apoB/Lp-PLA2 which are less well cleared from the circulation, while simvastatin under these inflammatory conditions reduces the secretion of Lp-PLA2 leading to the reduced formation of apoB/Lp-PLA2.
      The higher apoB/Lp-PLA2 levels may confer hypercholesterolemic patients with higher atherogenicity, because apoB/Lp-PLA2 may be more atherogenic compared with apoB/Lp-PLA2(−) particles. According to previously published data, the existence of Lp-PLA2 on LDL may influence its atherogenic potency by degrading the oxPLs formed during LDL oxidation and generating LPC (
      • Gonçalves I.
      • Edsfeldt A.
      • Ko N.Y.
      • Grufman H.
      • Berg K.
      • Björkbacka H.
      • Nitulescu M.
      • Persson A.
      • Nilsson M.
      • Prehn C.
      • et al.
      Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation.
      ), which is a central actor in the inflammatory reactions occurring during atherosclerotic plaque development and rupture. Indeed, LPC may contribute to the development of atherosclerotic plaques as well as to plaque vulnerability and rupture. This hypothesis is also supported by studies showing that the Lp-PLA2 and LPC content of human carotid plaques predict future cardiovascular events, and that the plaque content of Lp-PLA2 and LPC is increased in symptomatic human carotid plaques (
      • Herrmann J.
      • Mannheim D.
      • Wohlert C.
      • Versari D.
      • Meyer F.B.
      • McConnell J.P.
      • Gossl M.
      • Lerman L.O.
      • Lerman A.
      Expression of lipoprotein-associated phospholipase A(2) in carotid artery plaques predicts long-term cardiac outcome.
      ,
      • Mannheim D.
      • Herrmann J.
      • Versari D.
      • Gossl M.
      • Meyer F.B.
      • McConnell J.P.
      • Lerman L.O.
      • Lerman A.
      Enhanced expression of Lp-PLA2 and lysophosphatidylcholine in symptomatic carotid atherosclerotic plaques.
      ).
      In conclusion, by establishing an ELISA method to specifically determine the Lp-PLA2-bound apoB, we show for the first time that in patients with primary hypercholesterolemia the increase in apoB/Lp-PLA2 is relatively higher compared with apoB/Lp-PLA2(−). Furthermore, simvastatin therapy reduces these particles to a higher extent compared with apoB/Lp-PLA2(−), suggesting a role of Lp-PLA2 in the metabolism of apoB-containing lipoproteins. Because Lp-PLA2 plays an important proatherogenic role by degrading oxPLs formed during LDL oxidation and generating LPC, the predominance of apoB/Lp-PLA2 particles in the plasma of patients with primary hypercholesterolemia may be an important factor contributing to their higher atherogenicity and incidence of cardiovascular disease.

      Acknowledgments

      The authors thank the Atherothrombosis Research Centre of the University of Ioannina for providing access to the laboratory equipment and facilities. The reagents for Lp-PLA2 mass assay and the anti-Lp-PLA2 2C10 monoclonal antibody were kindly donated by diaDexus Inc., San Francisco, CA.

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