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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2236.)
© 2007 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Differential Effect of Hypolipidemic Drugs on Lipoprotein-Associated Phospholipase A₂

Vasilios G. Saougos; Afroditi P. Tambaki; Mihalis Kalogirou; Michael Kostapanos; Irene F. Gazi; Robert L. Wolfert; Moses Elisaf; Alexandros D. Tselepis

From the Department of Internal Medicine, Medical School (V.G.S., M.Ka., M.Ko., I.F.G., M.E.), and the Laboratory of Biochemistry, Department of Chemistry (A.P.T., A.D.T.), University of Ioannina, Greece; and dia Dexus Inc (R.L.W.), South San Francisco, Calif.

Correspondence to Prof Alexandros D. Tselepis, Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece. E-mail atselep{at}uoi.gr

	Abstract

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Objective— Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) is a predictor for incident atherosclerotic disease. We investigated the effect of 3 hypolipidemic drugs that exert their action through different mechanisms on plasma and lipoprotein-associated Lp-PLA₂ activity and mass.

Methods and Results— In 50 patients with Type IIA dyslipidemia were administered rosuvastatin (10 mg daily), whereas in 50 Type IIA dyslipidemic patients exhibiting intolerance to previous statin therapy were administered ezetimibe as monotherapy (10 mg daily). Fifty patients with Type IV dyslipidemia were given micronised fenofibrate (200 mg daily). Low- and high-density lipoprotein (LDL and HDL, respectively) subclass analysis was performed electrophoretically, whereas lipoprotein subfractions were isolated by ultracentrifugation. Ezetimibe reduced plasma Lp-PLA₂ activity and mass attributable to the reduction in plasma levels of all LDL subfractions. Rosuvastatin reduced enzyme activity and mass because of the decrease in plasma levels of all LDL subfractions and especially the Lp-PLA₂ on dense LDL subfraction (LDL-5). Fenofibrate preferentially reduced the Lp-PLA₂ activity and mass associated with the VLDL+IDL and LDL-5 subfractions. Among studied drugs only fenofibrate increased HDL-associated Lp-PLA₂ (HDL-Lp-PLA₂) activity and mass attributable to a preferential increase in Lp-PLA₂ associated with the HDL-3c subfraction.

Conclusion— Ezetimibe, rosuvastatin, and fenofibrate reduce Lp-PLA₂ activity and mass associated with the atherogenic apoB-lipoproteins. Furthermore, fenofibrate improves the enzyme specific activity on apoB-lipoproteins and induces the HDL-Lp-PLA₂. The clinical implications of these effects remain to be established.

We investigated the effect of ezetimibe, rosuvastatin, and fenofibrate on the lipoprotein-associated phospholipase A₂ (Lp-PLA₂) activity and mass, in hyperlipidemic patients. All drugs reduced Lp-PLA₂ activity and mass associated with the atherogenic apoB-lipoproteins, whereas fenofibrate was the only drug that improved the specific activity of the enzyme associated with these lipoproteins and significantly induced the HDL–Lp-PLA₂. The clinical implications of these effects remain to be established.

Key Words: hyperlipidemia • lipoproteins • PAF-acetylhydrolase • Lp-PLA₂ • ezetimibe • fenofibrate • rosuvastatin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Platelet-activating factor (PAF) acetylhydrolase exhibits a Ca²⁺-independent phospholipase A₂ activity and degrades PAF and oxidized phospholipids by catalyzing the hydrolysis of the ester bond at the sn-2 position.¹ PAF-acetylhydrolase in plasma is complexed to lipoproteins²; thus it is also referred as lipoprotein-associated phospholipase A₂ (Lp-PLA₂).³ Lp-PLA₂ is associated mainly with apolipoprotein B (apoB)-containing lipoproteins and primarily with low-density lipoprotein (LDL), whereas a small proportion of circulating enzyme activity is also associated with high-density lipoprotein (HDL).^1,2 The majority of the LDL-associated Lp-PLA₂ activity is bound to the atherogenic small-dense LDL (sdLDL) particles,^2,4,5 and we recently showed that the enzyme activity is a marker of sdLDL particles in plasma.⁶ Lp-PLA₂ is principally produced by hematopoietic cells including monocytes-macrophages.^7,8 Lp-PLA₂ has been identified in atherosclerotic plaques⁹; however, its role in atherosclerosis is still under investigation. In this regard, it is suggested that this enzyme might have an antiinflammatory role because it degrades and inactivates proinflammatory PAF and oxidized phospholipids^10,11; other studies showed that Lp-PLA₂ may have a proinflammatory and proatherogenic role¹² because it generates lysophosphatidylcholine (lysoPC)^3,13 and bioactive oxidized fatty residues.³

Data from large White population studies demonstrated an independent association between plasma Lp-PLA₂ with cardiovascular disease (CVD) risk. In this regard a recent metaanalysis showed that Lp-PLA₂ is significantly associated with CVD, and the risk estimate appears to be relatively unaffected by adjustment for conventional CVD risk factors.¹⁴ In contrast to total plasma enzyme, which mainly represents the LDL-associated Lp-PLA₂, several lines of evidence suggest that HDL-associated Lp-PLA₂ activity, although at low levels, contributes to the antiatherogenic effects of this lipoprotein.¹ However, the clinical value of HDL-associated Lp-PLA₂ as a potent inhibitor of the atherosclerotic process remains to be established.

Among the various agents used to treat patients with CVD, only drugs that affect lipid metabolism can significantly influence plasma Lp-PLA₂.^15,16 Thus several statins (atorvastatin, lovastatin, simvastatin, and fluvastatin) reduce the enzyme activity in plasma in parallel to a reduction in LDL-cholesterol levels.^17–21 In contrast, pravastatin increased plasma Lp-PLA₂ activity,²² whereas other investigators suggested that pravastatin reduced plasma Lp-PLA₂ mass.²³ Fibrates reduce plasma Lp-PLA₂ activity but significantly increase the HDL-associated Lp-PLA₂ activity.^16,24 These data suggest that there are significant differences on the effect of various hypolipidemic drugs on Lp-PLA₂ activity or mass in total plasma and in lipoprotein subspecies. To provide more insights into the effect of hypolipidemic drugs on plasma Lp-PLA₂, we investigated the effect of 3 agents that exert their action through different mechanisms (rosuvastatin, ezetimibe, and fenofibrate) on Lp-PLA₂ activity and mass in total plasma and in lipoprotein subspecies in hyperlipidemic patients.

	Materials and Methods

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Patients
One hundred and fifty hyperlipidemic patients participated in the present study. According to their lipid levels, patients were divided into the following groups: (1) primary hypercholesterolemia (type IIA dyslipidemia group, n=50, who were administered rosuvastatin), (2) type IIA dyslipidemia (n=50, who were administered ezetimibe), and (3) primary hypertriglyceridemia (type IV dyslipidemia, n=50, who were given micronized fenofibrate). Details on patient selection criteria, patient characteristics, and treatment are provided in the Data Supplement, available online at http://atvb.ahajournals.org.

Biochemical Parameters
Lipoprotein subclass analysis was performed electrophoretically by use of high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL System (Quantimetrix). Subfractionation of plasma lipoproteins by was performed by isopycnic density gradient ultracentrifugation. Lp-PLA₂ activity was measured by the trichloroacetic acid (TCA) precipitation procedure with the use of [³H]-PAF (100 µmol/L final concentration) as a substrate. Lp-PLA₂ mass was determined by use of a dual monoclonal antibody immunoassay standardized to recombinant Lp-PLA₂ (PLAC test; diaDexus, Inc). Data are presented as mean±SD, except for Lp(a), which was expressed as the median and range. Statistical analysis was performed using SPSS 13.0 softpack. Details on the methodology used and statistical analysis are provided in the Data Supplement, available online at http://atvb.ahajournals.org.

	Results

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Effect of Hypolipidemic Therapy on Serum Lipid Profile
As shown in supplemental Table I (available online at http://atvb.ahajournals.org), ezetimibe significantly decreased serum total cholesterol, LDL-cholesterol, non–HDL-cholesterol, and apoB levels. Furthermore, ezetimibe induced a slight but significant reduction in serum HDL-cholesterol levels whereas it did not affect apoA-I levels. Rosuvastatin significantly decreased serum triglycerides, total cholesterol, LDL-cholesterol, non–HDL-cholesterol, and apoB levels. The reduction in these serum lipid parameters was more profound compared with the ezetimibe group. Fenofibrate significantly decreased serum levels of triglycerides, total cholesterol, LDL-cholesterol, non–HDL-cholesterol, and apoB. Furthermore, fenofibrate induced a significant increase in the serum levels of HDL-cholesterol and apoA-I, a phenomenon not observed in the other 2 groups. Finally, no alterations in the baseline Lp(a) levels were induced by any lipid-lowering therapy (supplemental Table I).

Effect of Hypolipidemic Therapy on Lipoprotein Subclasses
Type IV dyslipidemic patients had higher plasma levels of VLDL-cholesterol and lower levels of IDL-cholesterol and buoyant LDL-cholesterol at baseline compared with type IIA dyslipidemic patients of either the ezetimibe or the rosuvastatin group. Type IV dyslipidemic patients had significantly higher baseline levels of sdLDL-cholesterol and a higher proportion of sdLDL whereas the mean LDL size was lower compared with type IIA patients. Treatment with either ezetimibe or rosuvastatin significantly reduced the mass of all apoB-containing lipoprotein subclasses, with the exception of VLDL-cholesterol, which was not reduced by ezetimibe. However, neither drug affected sdLDL proportion and mean LDL size. Fenofibrate significantly reduced VLDL-cholesterol levels but it did not affect IDL-cholesterol or buoyant LDL-cholesterol levels. Finally, fenofibrate reduced sdLDL-cholesterol levels (and therefore the proportion of sdLDL) and increased mean LDL size (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effect of Hypolipidemic Drugs on ApoB- and apoA-I–Containing Lipoprotein Subclasses

Type IV dyslipidemic patients had lower levels of HDL-2 and HDL-3 subclasses at baseline compared with type IIA patients of either ezetimibe or rosuvastatin group. Ezetimibe significantly reduced the concentration of small HDL-3 subclass without affecting the concentrations of the large HDL-2 subclass. In contrast, rosuvastatin did not affect the mass of either HDL subclass. Finally, fenofibrate significantly increased the mass of both HDL subclasses (Table 1).

Plasma Lp-PLA₂ Activity and Mass
Ezetimibe significantly decreased total plasma and non-HDL–Lp-PLA₂ activity and mass (Table 2); however, it did not affect the enzyme specific activity or the Lp-PLA₂ activity to apoB ratio (in nmol/mg/min, 0.58±0.13 before versus 0.60±0.15 posttreatment) and the Lp-PLA₂ mass to apoB ratio (in ng/mg, 3.75±0.51 before versus 3.83±0.98 posttreatment). Rosuvastatin significantly reduced total plasma and non-HDL Lp-PLA₂ activity and mass; these reductions were more pronounced compared with those induced by ezetimibe (Table 2). Like ezetimibe, rosuvastatin did not alter the enzyme specific activity or the ratios of Lp-PLA₂ activity or mass to apoB. Fenofibrate significantly reduced total plasma and non-HDL Lp-PLA₂ activity. Importantly, fenofibrate induced a significant increase in the total plasma and non-HDL enzyme specific activity (Table 2). Baseline and posttreatment corrrelations between non-HDL–Lp-PLA₂ activity and mass are shown in Figure 1.

View this table: [in this window] [in a new window]   Table 2. Effect of Hypolipidemic Drugs on Plasma and Lipoprotein-Associated Lp-PLA2 Activity, Mass, and Specific Activity

View larger version (16K): [in this window] [in a new window]   Figure 1. Correlation between non-HDL–Lp-PLA2 mass and activity at baseline and after treatment with ezetimibe, rosuvastatin, or fenofibrate.

Ezetimibe induced a slight but significant reduction in HDL–Lp-PLA₂ activity and mass but it did not affect the enzyme specific activity or the HDL-Lp-PLA₂ activity to apoA-I ratio (in nmol/mg/min, 0.021±0.005 before versus 0.021±0.006 posttreatment) and the HDL-Lp-PLA₂ mass to apoA-I ratio (in ng/mg, 0.52±0.17 before versus 0.49±0.18 posttreatment). By contrast rosuvastatin did not affect either of the above parameters.

Importantly, fenofibrate increased HDL-Lp-PLA₂ activity and mass but it did not affect the enzyme specific activity (Table 2). Furthermore, fenofibrate significantly increased the ratios of HDL-Lp-PLA₂ activity to apoA-I (in nmol/mg/min, 0.019±0.008 before versus 0.030±0.009 posttreatment, P<0.03) or HDL-Lp-PLA₂ mass to apoA-I (in ng/mg, 0.48±0.16 before versus 0.69±0.21 posttreatment P<0.03).

Lp-PLA₂ Activity and Mass on Lipoprotein Subfractions
We further investigated the effect of hypolipidemic drugs on the Lp-PLA₂ activity and mass associated with the apoB- and apoA-I-containing lipoprotein subspecies, isolated by density gradient ultracentrifugation. Among the apoB-containing lipoproteins, Lp-PLA₂ activity and mass were preferentially associated with the dense LDL-5 subfraction in all patient groups at baseline (Figure 2A and 2B), a finding that is in accordance to our previous results.^6,17,24 Ezetimibe significantly reduced the enzyme activity and mass (expressed per mL of plasma) associated with all apoB-containing lipoprotein subfractions (Figure 2A and 2B), whereas it did not affect the enzyme activity or mass when it was expressed per mg of protein (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 2. Bar graphs illustrating the effect of ezetimibe and rosuvastatin on the Lp-PLA2 activity (A), Lp-PLA2 mass (B), and Lp-PLA2 specific activity (C) associated with apoB-containing lipoprotein subspecies in patients with Type IIA dyslipidemia. Lipoprotein subspecies were fractionated by isopycnic density gradient ultracentrifugation of plasma. Enzymatic activity was determined by the trichloroacetic acid (TCA) precipitation procedure and enzyme mass by use of a dual monoclonal antibody immunoassay. Lp-PLA2 specific activity was expressed as a ratio of the enzyme activity to the enzyme mass. Values represent the mean±SD. *P<0.01 compared with respective baseline values.

Rosuvastatin reduced the enzyme activity and mass (expressed per mL of plasma) associated with all apoB-containing lipoprotein subfractions (Figure 2A and 2B). Remarkably, it significantly reduced the enzyme activity and mass (expressed per mg of protein) in the dense LDL-5 subfraction (Lp-PLA₂ activity, nmol/mg/min: 38±13 at baseline versus 20±9 posttreatment, P<0.03; Lp-PLA₂ mass, ng/mg: 95±29 at baseline versus 50±18 posttreatment, P<0.03). Neither ezetimibe nor rosuvastatin affected the enzyme specific activity of each apoB lipoprotein subfraction (Figure 2C). It should be noted that the Lp-PLA₂ specific activity at baseline in apoB-containing lipoprotein subfractions ranges from 0.3 (LDL-5) to 2.3 nmol/ng/min (LDL-3) (Figure 2) whereas the non-HDL enzyme specific activity in the same patient groups is lower and ranges from 0.12 to 0.22 nmol/ng/min (Table 2). However, when the enzyme specific activity in LDL-5 and LDL-3 was determined in the presence of human serum albumin or total plasma proteins (prepared as described in the methods section), it was reduced in a dose-dependent manner. Thus at an albumin or total plasma protein concentration of 6 g/dL a significant reduction of 50±8% in both subfractions for both treatments was observed, suggesting that plasma proteins, primarily albumin, significantly affect the enzyme activity a finding, which is in accordance with previously published results.²⁵ Thus the Lp-PLA₂ specific activity in LDL-5 in the presence of 6 g/dL albumin is 0.15±0.02 nmol/ng/min being similar to that of the non-HDL–Lp-PLA₂, a finding consistent with the preferential association of the non-HDL enzyme with LDL-5.

Fenofibrate significantly reduced the Lp-PLA₂ activity and mass associated with the VLDL+IDL subfraction (Figure 3A and 3B), whereas it did not affect the enzyme-specific activity of this subfraction (Figure 3C). Fenofibrate did not affect the activity, mass, or specific activity of the enzyme associated with large and intermediate LDL particles (LDL-1 to LDL-4); however, it significantly reduced the enzyme activity and mass associated with LDL-5 (Figure 3A and 3B). Furthermore, fenofibrate induced a significant increase in the specific activity of Lp-PLA₂ associated with this subfraction (Figure 3C).

View larger version (10K): [in this window] [in a new window]   Figure 3. Bar graphs illustrating the effect of fenofibrate therapy on the Lp-PLA2 activity (A), Lp-PLA2 mass (B), and Lp-PLA2 specific activity (C) associated with VLDL+IDL and dense LDL-5 subspecies in patients with Type IV dyslipidemia. Lipoprotein subspecies were fractionated by isopycnic density gradient ultracentrifugation of plasma. Enzymatic activity was determined by the trichloroacetic acid (TCA) precipitation procedure and enzyme mass by use of a dual monoclonal antibody immunoassay. Lp-PLA2 specific activity was expressed as a ratio of the enzyme activity to the enzyme mass. Values represent the mean±SD. *P<0.001 and **P<0.01 compared with respective baseline values.

Finally, it should be noted that no detectable amounts of Lp(a) were found in any lipoprotein subfraction at baseline or after treatment with any hypolipidemic drug. Thus it is unlikely that the alterations in the Lp-PLA₂ associated with LDL subfractions induced by hypolipidemic therapy are influenced by changes in the Lp(a) levels and in the Lp(a)-associated Lp-PLA₂.

Among the HDL subfractions, Lp-PLA₂ activity and mass in all patient groups at baseline were preferentially associated with HDL-3c, a finding which is in accordance with our previously published results.^6,17,24 Ezetimibe slightly, albeit significantly, reduced Lp-PLA₂ activity and mass associated with the HDL-3c subfraction, a phenomenon not observed after rosuvastatin administration. By contrast, fenofibrate significantly increased Lp-PLA₂ activity and mass associated with HDL-3c (Figure 4A and 4B). Neither drug influenced the activity or mass of Lp-PLA₂ associated with the other HDL subfractions. Finally, neither drug affected the enzyme specific activity on any HDL subfraction (in nmol/ng/min, 0.32±0.12 for HDL-2b, 0.24±0.10 for HDL-2a, 0.12±0.04 for HDL-3a, 0.07±0.02 for HDL-3b, and 0.04±0.01 for HDL-3c).

View larger version (14K): [in this window] [in a new window]   Figure 4. Bar graphs illustrating the effect of ezetimibe, rosuvastatin, and fenofibrate on the Lp-PLA2 activity (A) and Lp-PLA2 mass (B) associated with HDL-3c subfraction. HDL subfractions were isolated by isopycnic gradient ultracentrifugation of plasma depleted of apoB-lipoproteins. Enzymatic activity was determined by the trichloroacetic acid (TCA) precipitation procedure and enzyme mass by use of a dual monoclonal antibody immunoassay. Values represent the mean±SD. *P<0.01 compared with respective baseline values.

It should be emphasized that the mean value of enzyme specific activity in total HDL formed by mixing of equal volumes of all HDL subfractions (0.15 nmol/ng/min), is much lower compared with that of total LDL formed by mixing of equal volumes of the subfractions LDL-1 to LDL-5 (1.34 nmol/ng/min). Importantly, when both lipoproteins were dissociated by treatment with 0.1% Triton X-100, the Lp-PLA₂ specific activity on LDL was significantly reduced to 0.93 nmol/ng/min (P<0.03) because of the increase by 36% in the enzyme mass (from 23.8±4.2 to 32.4±5.1 ng/mg of total protein, P<0.03). Neither the enzyme activity in both lipoproteins nor the enzyme mass in HDL was significantly influenced by this treatment. These results show that the method used for the determination of Lp-PLA₂ mass may not detect all active enzyme in LDL, a phenomenon not observed for HDL.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study compares for the first time the effect of 3 hypolipidemic drugs (ezetimibe, rosuvastatin, fenofibrate), which exert their action through different mechanisms, on plasma Lp-PLA₂ activity and mass. All drugs reduce Lp-PLA₂ activity and mass associated with the atherogenic apoB-containing lipoproteins. Furthermore, fenofibrate increases the specific activity of the enzyme associated with these lipoproteins and specifically that of the most atherogenic dense LDL-5 subfraction.

Fenofibrate reduces Lp-PLA₂ activity and mass associated with apoB-containing lipoproteins, an effect that could be mainly attributed to the preferential reduction of the enzyme associated with LDL-5 particles,²⁴ ie, those particles carrying the majority of LDL-associated enzyme.^2,6 In accordance with our previously published results,²⁴ the present study shows that the above reduction is attributed to the fenofibrate action to decrease sdLDL and to increase large buoyant LDL particles, which have a higher clearance rate than sdLDL.²⁶ A contributory role to the reduction of Lp-PLA₂ by fenofibrate plays also the decrease in enzyme associated with the triglyceride-rich VLDL+IDL subfraction (attributable to the drug-induced reduction in the plasma concentration of this subfraction).

An important observation of the present study is that the non-HDL–Lp-PLA₂ specific activity is significantly increased by fenofibrate. According to our previous results, the specific activity of Lp-PLA₂ associated with large buoyant LDL is higher than that of either LDL-5 or VLDL+IDL subfraction.⁶ In this regard, the results of the present study show that the method used for the determination of Lp-PLA₂ mass may not detect all active enzyme in LDL, a phenomenon not observed for HDL, suggesting that structural differences among lipoprotein species may significantly influence the determination of enzyme mass, a hypothesis that needs further investigation. Based on the above observations, we may suggest that the increase in non-HDL–Lp-PLA₂ specific activity by fenofibrate is attributed to the drug-induced preferential reduction in the enzyme associated with LDL-5 and VLDL+IDL subfractions. A contributory role in the above phenomenon may also play the fenofibrate-induced increase in the specific activity of Lp-PLA₂ associated with LDL-5.

In accordance with our previously published results,²⁴ fenofibrate treatment increases the HDL–Lp-PLA₂ activity. It also increases the HDL–Lp-PLA₂ mass, thus it does not affect the enzyme specific activity. This effect is attributable to the drug-induced increase in plasma levels of both HDL-2 and HDL-3 subspecies as well as to the preferential enrichment of the HDL-3c in Lp-PLA₂. We had previously suggested that the latter phenomenon is attributed to enzyme transfer from triglyceride-rich apoB-containing lipoproteins to HDL during their enhanced lipolysis by lipoprotein lipase induced by fenofibrate.^24,27 Although the role of the HDL-Lp-PLA₂ in humans has not been established yet, data from in vitro experiments as well as in vivo studies in animal models suggest that this enzyme may significantly contribute to the antiatherogenic effects of HDL (reviewed in¹). Consequently, the increase of HDL-Lp-PLA₂ induced by fenofibrate may represent an important antiatherogenic effect of this drug, a hypothesis that needs further investigation.

The present study further demonstrates that the administration of rosuvastatin in type IIA dyslipidemic patients significantly reduces Lp-PLA₂ activity and mass associated with apoB-containing lipoproteins. This reduction is the highest observed among all statins used in previous studies,^16–23 and it could be primarily attributed to the drug-induced reduction in the plasma concentration of all LDL subfractions and to the preferential reduction of Lp-PLA₂ associated with LDL-5. It has been suggested that simvastatin reduces LDL-associated Lp-PLA₂ not only through the receptor-mediated removal of LDL but also through a receptor-independent clearance of the lipid and enzyme contents of LDL.²⁰ This mechanism may explain our results on the preferential reduction in Lp-PLA₂ associated with LDL-5 (expressed per mg of protein) induced by rosuvastatin.

Ezetimibe is a drug that acts by inhibiting the absorption of cholesterol at the brush border of the intestinal wall.^28,29 The present study shows for the first time that it reduces the plasma levels of Lp-PLA₂ mass and activity (although to a lesser extent compared with rosuvastatin and fenofibrate), by reducing the plasma concentration of all apoB-containing lipoprotein subfractions. Furthermore, ezetimibe induces a slight but significant decrease in the HDL-Lp-PLA₂ activity and mass. This follows the lowering effect of ezetimibe on plasma HDL-cholesterol levels a finding, which is not consistent in all studies³⁰ and may reflect the relatively high pretreatment levels of HDL-cholesterol in our population resulting in a regression to the mean effect. Because ezetimibe does not influence the ratio of enzyme activity or mass to apoA-I levels, we suggest that the reduction in HDL-3 plasma concentration may account for the ezetimibe-induced decrease in the HDL-Lp-PLA₂. This is further supported by the finding that ezetimibe decreases the enzyme associated only with HDL-3c subfraction. It should be noted that all patients treated with ezetimibe were statin-intolerant, therefore the above results may not be representative of other groups given ezetimibe.

In addition to LDL and HDL, another carrier of Lp-PLA₂ in plasma is Lp(a). Interestingly, we³¹ and others³² have demonstrated that Lp(a) is enriched in Lp-PLA₂ compared with LDL. However, Lp(a) can influence the distribution of Lp-PLA₂ between LDL and HDL in plasma only when its plasma levels exceed 8 mg/dL.³¹ Thus, it is unlikely that the Lp(a)-associated Lp-PLA₂ could influence the enzyme changes induced by hypolipidemic drugs, because the present results showed that no alterations in Lp(a) levels were induced by any lipid-lowering therapy and no patient exhibited a baseline or posttreatment Lp(a) levels above 8 mg/dL. Finally, no detectable amounts of Lp(a) were found in any lipoprotein subfraction at baseline or after treatment with any hypolipidemic drug.

Clinical studies have shown an independent association between plasma levels of Lp-PLA₂ mass or activity and CVD.¹⁴ The present study further shows that the determination of Lp-PLA₂ mass, activity, and specific activity on individual lipoprotein subfractions may be pathophysiologically and clinically important. In this regard, we have previously shown that the preferential enzyme distribution on sdLDL particles compared with large buoyant apoB-containing has as a consequence an increased production of lysoPC, the main metabolite of Lp-PLA₂, during oxidation of this subfraction.¹³ Several studies have supported the important role of lysoPC in atherogenesis,³³ and more recently it was shown that local coronary production of lysoPC is associated with endothelial dysfunction and early atherosclerosis.³⁴ Furthermore, the preferential association of Lp-PLA₂ with HDL-3 subfraction as compared with other apoA-I–containing lipoproteins may contribute to the antiinflammatory and antioxidant effects of these particles.¹ Finally the Lp(a)-associated Lp-PLA₂ may play an important role in the metabolism of oxidized phospholipids in humans, in view of emerging data showing that oxidized phospholipids in plasma are preferentially sequestered on Lp(a).³⁵ Taking into account our previous results showing that the type of dyslipidemia and the underlying metabolic defect significantly influence the enzyme distribution among lipoprotein subspecies,^5,17,24 we suggest that the determination of enzyme parameters on specific lipoprotein subspecies may provide useful information on both pathophysiological and clinical basis, in addition to the valuable information provided from the measurement of Lp-PLA₂ mass and activity in total plasma.

A limitation of the present study could be the selection of patients who were allocated to different therapeutic agents according to the NCEP ATPIII goals that resulted in populations with different types of dyslipidemias. Thus it must be acknowledged that the different lipid abnormalities observed between Type IIA and Type IV patient groups may have contributed to the differential effect on the lipoprotein-associated Lp-PLA₂ levels of fenofibrate (administered in Type IV patients) compared with rosuvastatin or ezetimibe (given to Type IIA individuals).

In conclusion the present study demonstrates for the first time that fenofibrate, rosuvastatin, and ezetimibe (acting through different mechanisms) reduce Lp-PLA₂ activity and mass associated with the atherogenic apoB-containing lipoproteins, the rosuvastatin exhibiting the most potent effect. Additionally, fenofibrate increases the specific activity of the non-HDL–Lp-PLA₂ as well as the HDL–Lp-PLA₂ mass and activity. The clinical implications of these effects remain to be established.

	Acknowledgments

 
We are grateful to Dr Konstantinos G. Lagos and Dr Eleni S. Nakou for skilled technical assistance.

Sources of Funding

This work was supported by research grants from the Greek Ministry of Research and Technology (PENED program 2003 ED 643).

Disclosures

None.

	Footnotes

 
Original received September 19, 2006; final version accepted July 4, 2007.

	References

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