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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 10 5021-5029
Copyright © 2004 by The Endocrine Society

Effects of Atorvastatin on Fasting Plasma and Marginated Apolipoproteins B48 and B100 in Large, Triglyceride-Rich Lipoproteins in Familial Combined Hyperlipidemia

C. Verseyden, S. Meijssen and M. Castro Cabezas

Departments of Internal Medicine, University Medical Center Utrecht (C.V., S.M., M.C.C.), 3508 GA Utrecht, The Netherlands; Sint Franciscus Gasthuis Rotterdam (M.C.C.), 3045 PM Rotterdam, The Netherlands; and Department of Nephrology, Leiden University Medical Center (C.V.), 2333 ZA Leiden, The Netherlands

Address all correspondence and requests for reprints to: Dr. M. Castro Cabezas, Department of Internal Medicine F02.126, University Medical Center Utrecht, Heidelberglaan 100, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. E-mail: m.castrocabezas{at}azu.nl.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Large triglyceride (TG)-rich lipoproteins (TRLs) circulate in the blood, but they may also be present in a marginated pool, probably attached to the endothelium. It is unknown whether statins can influence this marginated pool in vivo in humans. Intravenous fat tests were performed in familial combined hyperlipidemia (FCHL) subjects before and after atorvastatin treatment and in controls to investigate whether acute increases in apoB in TRL fractions would occur, potentially reflecting the release of this TRL from a marginated pool. After a 12-h fast, a bolus injection of 10% Intralipid was given to 12 FCHL patients before and after 16-wk treatment with atorvastatin. Twelve carefully matched controls were included. For 60 min postinjection, apoB48, apoB100, and lipids were measured in TRLs. Fasting apoB100 in all TRL fractions were 2- to 3-fold higher in untreated FCHL compared with controls. ApoB48 concentrations in chylomicron fractions increased significantly within 10 min in FCHL before and after treatment, but not in controls. ApoB100 increased significantly in the chylomicron fractions in untreated FCHL and in controls, but not in FCHL after treatment. In very low density lipoprotein 1, apoB100 increased only in untreated FCHL. In very low density lipoprotein 2, apoB100 did not change in any group. These data show that increasing the number of circulating TRLs by chylomicron-like particles, results in increased plasma apoB-TRLs, probably by acute release from a marginated pool. This is a physiological process occurring in FCHL and in healthy normolipidemic subjects, but it is more pronounced in the former. Decreased marginated TRL particles in FCHL is a novel antiatherogenic property of atorvastatin.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
TRIGLYCERIDES (TGs) ARE transported in plasma incorporated in different lipoproteins. In the small intestine, TG are derived from dietary fat and incorporated into chylomicrons, which contain one molecule of apolipoprotein (apo) B48 as a structural protein in each particle (1, 2). The other large TG-rich lipoproteins (TRLs), very low density lipoproteins (VLDLs), are only secreted by the liver in humans (1) and contain one apoB100 molecule per lipoprotein (1, 2, 3). Therefore, measurement of apoB reflects the number of circulating lipoproteins (4, 5, 6).

TRLs and their remnants are cleared by the liver by low density lipoprotein (LDL) receptors and remnant receptors (7, 8, 9). In extrahepatic tissues, other receptors are involved (10, 11, 12). In vitro experiments have shown that lipoprotein lipase (LPL) also serves as a ligand for binding of lipoproteins to cell surfaces, thereby mediating lipoprotein clearance (13, 14, 15). The binding of TRLs attached to the endothelium by LPL can be disrupted by free fatty acids (FFA) (16, 17). TRLs and their remnants can also be marginated, and they remain attached to the endothelium in vivo as has been shown in animals and humans (18, 19). Karpe et al. (19) showed that margination in healthy males is more pronounced for chylomicrons and VLDL1. Marginated lipoproteins most likely are bound to the endothelium by LPL-phospholipid interactions or by means of receptor-ligand interactions in different tissues and represent those lipoproteins that potentially are in the process of migrating into the subendothelial space (20).

If margination occurs in humans, it is still unknown whether it can be modulated. In this study we included untreated FCHL patients as a model to investigate TRL margination. FCHL is characterized by VLDL overproduction and delayed remnant clearance (21, 22, 23), and this combination of abnormalities could lead to a greater extent of margination. The untreated FCHL were compared with carefully matched, healthy controls. In addition, the effects of atorvastatin in FCHL were evaluated.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The independent ethics committee of University Medical Center Utrecht approved the study protocol, and written informed consent was obtained from each participant. Twelve unrelated FCHL patients fulfilled the following criteria: they were known to have primary hyperlipidemia with varying phenotypic expression and had elevated plasma apoB concentrations (>1.20 g/liter) when untreated, they had at least one first degree relative had a different hyperlipidemic phenotype; and each index subject had a positive family history of premature coronary heart disease, defined as myocardial infarction or cerebrovascular disease before the age of 60 yr. In addition, the patients fulfilled the following inclusion criteria: absence of xanthomas and secondary factors associated with hyperlipidemia, body mass index (BMI) less than 30 kg/m2, absence of apo E2/E2 genotype, and use of no more than 3 U alcohol/d.

Twelve normolipidemic, healthy volunteers without a family history of cardiovascular disease, absence of apo E2/E2 genotype, BMI less than 30 kg/m2, use of no more than 3 U/alcohol/d, and not using drugs known to affect lipid metabolism were recruited by advertisement. Controls were matched to FCHL patients by age, waist to hip ratio, and BMI.

Treatment period

At inclusion, five FCHL patients were using lipid-lowering drugs (atorvastatin, 10, 40, or 80 mg once daily; simvastatin, 20 mg once daily; fluvastatin, 20 mg once daily). They stopped their medication 4 wk before the first iv fat-loading test.

FCHL patients were treated for 16 wk with atorvastatin. The initial dose of atorvastatin was 10 mg once daily. Every 4 wk, the patients visited the out-patient clinic, and fasting plasma lipids and apolipo-proteins were measured. When plasma TG concentrations were above 2 mM and/or cholesterol levels were above 6.5 mM, the atorvastatin dose was doubled, up to a maximum dose of 80 mg after 12 wk. After 16 wk of atorvastatin, a second iv fat-loading test was performed. Control subjects received no treatment and underwent the iv fat-loading test once.

Intravenous fat-loading test

Intralipid (10%; Pharmacia-Upjohn, Uppsala, Sweden) was used as a fat source; this emulsion is to some extent chylomicron-like, because its lipid droplets have a size and metabolic fate similar to those of chylomicrons. However, the emulsion does not contain cholesterol or apoB. After an overnight fasting period of 12 h, Intralipid was given in a bolus of 0.1 g/kg body weight in one arm. Peripheral blood samples were obtained in sodium EDTA (2 mg/ml) before (–5 and 0 min) and at 1, 2, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min after the bolus injection from the other arm.

Analytical methods and laboratory techniques

Blood was placed on ice and centrifuged immediately for 15 min at 800 x g at 4 C. After centrifugation, a protease inhibitor was added to the plasma as previously described (6). Tetrahydrolipstatin (Roche, Basel, Switzerland) was added to plasma that was used for FFA measurements to a final concentration of 1 mg/liter to prevent in vitro lipolysis (24). Plasma samples were stored at –20 C immediately after centrifugation. ApoE genotypes were determined as described previously (6, 21). TG and cholesterol were measured in duplicate by a commercial colorimetric assay (GPO-PAP and CHOD-PAP, respectively, Roche, Indianapolis, IN). High density lipoprotein cholesterol was determined as described by Burstein et al. (25) using the phosphotungstate/MgCl2 method. Lipoproteins at 0, 5, 10, 20, 40, and 60 min were subfractionated by ultracentrifugation as previously described in detail (6). Briefly, a discontinuous density gradient ranging from a density of 1.10 g/ml (adjusted plasma), followed by densities of 1.063, 1.019, and 1.006 g/ml potassium bromide solutions, was formed in Ultraclear tubes (Beckman Instruments, Inc, Palo Alto, CA; 14 x 95 mm). Ultracentrifugation was performed in a Beckman SW40 Ti bucket rotor at 40,000 rpm at 4 C in a Beckman LE-80 ultracentrifuge. Consecutive runs were carried out to float Svedberg flotation (Sf) more than 400 (32 min), Sf 60–400 (3 h, 28 min), and Sf 20–60 (17 h) (6). These fractions correspond to chylomicrons, VLDL1 and VLDL2, respectively. Plasma and lipoprotein fractions were always kept at 4 C.

ApoB48 and apoB100 in TRLs (chylomicrons, VLDL1, and VLDL2) were quantitated by SDS-PAGE (5, 6). Briefly, samples of each TRL fraction were delipidated in a methanol/diethyl ether solvent system. After the first step, debris was removed with ice-cold diethyl ether, and the proteins were dried by vaporization. The material was dissolved in sample buffer. Aliquots for apoB determination were stored at –80 C and assayed within 3 months on 3–5% SDS-PAGE. The amount of apoB100 in the TRL fractions is usually too high to quantitate directly by SDS-PAGE; therefore, each sample was diluted 20 times with sample buffer and then loaded on the gel. For quantitation of apoB48, each sample was loaded on the gel undiluted. The standard curve was made by delipidated LDL with known absolute amounts of proteins. To assess the equality of chromogenicities of apoB48 and apoB100, human chylous ascites containing significant amounts of apoB48 was also delipidated and run on each gel (6). The proteins were stained with a Colloidal Blue staining kit from Novex (Invitrogen Life Technologies, Carlsbad, CA), containing Coomassie G-250, and were destained by washing the gels at least four times with distilled water. For quantitation of apoB48 and apoB100, a personal computer-based image analysis system was used (6). Quantitation of apoB48 and apoB100 was performed by a technician who was blinded to the code of the samples. The reproducibility of the method, calculated as 100% minus the coefficient of variation, was 92.7%. The recovery of the apoB samples ranged from 65–90%. ApoAI was measured by nephelometry using apoAI polyclonal antibodies (OUED 14/15; Behring Diagnostics NV, Leusden, The Netherlands). FFA were measured by a commercial enzymatic colorimetric assay (Wako Chemicals, Neuss, Germany).

Statistical methods

The primary aim of our study was to quantitate the amount of apoB-containing TRLs acutely released into the circulation after injection with Intralipid, potentially reflecting marginated TRLs. The secondary aim was to investigate the effects of atorvastatin on this acute apoB increases in FCHL.

All values in the text and tables are expressed as the mean ± SD. In the figures, the mean ± SEM are used. Mean differences between FCHL subjects and controls were calculated by independent sample t test. Mean differences between untreated and treated FCHL subjects were calculated by paired sample t test. Differences in fasting TG values between FCHL and controls were calculated by the Mann-Whitney test. Changes in time were calculated by repeated measures ANOVA with the least significant difference as the post hoc test compared with the baseline concentration at 0 min, with Bonferroni correction for multiple comparisons. To evaluate the acute changes in apoB48 and apoB100 after injection with Intralipid, areas under the curve (AUCs) and incremental AUCs are calculated for the first 10 min of each curve. Statistical calculations were performed using SPSS 10.0 (SPSS, Inc., Chicago, IL). Statistical significance was reached at P < 0.05 (two-tailed test).


   Results
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 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
General characteristics and treatment effects on fasting measurements (Table 1Go)

FCHL patients and matched controls had similar anthropometric characteristics according to the inclusion criteria. Fasting plasma concentrations of TG, cholesterol, and apoB were significantly higher in untreated FCHL patients compared with controls. Fasting apoAI was significantly lower in FCHL. Fasting plasma FFA concentrations and high density lipoprotein cholesterol were similar in both groups (Table 1Go).


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  		TABLE 1. The anthropometric characteristics and fasting values of metabolic parameters of 12 FCHL subjects before and after atorvastatin treatment and 12 controls

 
After 16 wk, at the end of the titration period, two patients used 10 mg, five patients used 20 mg, one patient used 40 mg, and four patients used 80 mg atorvastatin. Plasma cholesterol, LDL cholesterol, and apoB concentrations decreased significantly after atorvastatin treatment. Plasma TG remained elevated in FCHL compared with controls (P < 0.001).

Plasma TG, FFA, and cholesterol concentrations after Intralipid (Fig. 1Go)

In both groups, maximal TG concentrations were reached 1 min after the bolus injection of Intralipid (P < 0.001), followed by a gradual decline (Fig. 1AGo).



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  		FIG. 1. Mean (±SEM) plasma changes during the iv fat load of TGs (A), FFA (B), and cholesterol (C) in FCHL subjects before (•) and after ({circ}) atorvastatin treatment and in controls ({triangleup}). Significant differences compared with baseline are indicated by a circle around the symbol.

 
The injection of Intralipid resulted in a similar plasma FFA response. FFAs increased to a peak after 1 min (P < 0.001). After 60 min, FFA levels were still elevated in all groups compared with fasting concentrations (P < 0.05; Fig. 1BGo).

A small, but significant, plasma cholesterol decrease was observed at 2 min after the injection in FCHL patients before (5.58 ± 1.13 mM) and after (4.13 ± 1.46 mM) atorvastatin treatment (P < 0.05 for each), but not in controls (Fig. 1CGo).

TG and cholesterol concentrations in TRL fractions after Intralipid (Fig. 2Go and Table 2Go)

Fasting TG in all TRLs before and after atorvastatin treatment were elevated compared with control levels (Table 2Go). Chylo-TG concentrations increased significantly to a similar peak at 5 min in all groups (untreated and treated FCHL and controls; Fig. 2AGo). VLDL1 TG increased significantly in untreated FCHL to 0.81 ± 0.51 mM (P < 0.05) and in controls to 0.37 ± 0.15 mM (P < 0.05), but not in treated FCHL. VLDL2 TG in FCHL and controls did not change.



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  		FIG. 2. Mean (±SEM) changes during the iv fat load of TGs in chylo (A), VLDL1 (B), and VLDL2 (C) fractions and of cholesterol in chylo (D), VLDL1 (E), and VLDL2 (F) fractions in FCHL subjects before (•) and after ({circ}) atorvastatin and in controls ({triangleup}). Note that the scale of the y-axis differs in each panel. Significant differences compared with baseline are indicated by a circle around the symbol.

 

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  		TABLE 2. Fasting values of TG, cholesterol, apoB48, and apoB100 in three TRL fractions in 12 FCHL subjects before and after atorvastatin treatment and in 12 controls

 
Fasting cholesterol levels in untreated FCHL were significantly elevated in all TRL fractions compared with control levels. Chylo-cholesterol increased significantly to a maximum peak at 5 min in untreated FCHL of 0.16 ± 0.13 mM, in treated FCHL to 0.17 ± 0.14 mM, and in controls to 0.14 ± 0.11 mM (P < 0.01, for each). VLDL1 cholesterol decreased initially in untreated FCHL, followed by an increase to a maximum at 40 min (P < 0.001). In treated FCHL and controls, VLDL1 cholesterol increased to a peak at 40–60 min (P < 0.001). In VLDL2, cholesterol concentrations decreased in untreated and treated FCHL subjects and controls (P < 0.05; Fig. 2FGo).

ApoB48 and apoB100 concentrations in TRL fractions in FCHL and controls after Intralipid (Fig. 3Go and Table 2Go)

Fasting apoB48 did not differ between untreated FCHL patients and controls (Table 2Go). Injection of Intralipid resulted in a significant increase in apoB48 only in the chylo fraction in treated FCHL (from 0–10 min; P < 0.05), and a trend was observed in untreated FCHL (P = 0.080) and controls (from 0–20 min; P = 0.05; Fig. 3AGo). In the VLDL1 fraction, apoB48 concentrations remained unchanged compared with baseline in untreated FCHL and controls in the first 20 min after Intralipid injection. Atorvastatin had no effect on the apoB48 response. VLDL2-apoB48 did not change during the iv fat load in any group.



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  		FIG. 3. Mean (±SEM) changes during the iv fat load of apoB48 in chylo (A), VLDL1 (B), and VLDL2 (C) fractions and of apoB100 in chylo (D), VLDL1 (E), and VLDL2 (F) fractions in untreated (•) and treated ({circ}) FCHL patients and controls ({triangleup}). Note that the scale of the y-axis differs in each panel. Significant differences compared with baseline are indicated by a circle around the symbol.

 
Mean fasting apoB100 concentrations were significantly higher in untreated FCHL compared with controls in all fractions (Table 2Go). No differences were observed in fasting apoB100 after treatment in FCHL patients. VLDL1 apoB100 and VLDL2 apoB100 were still elevated in FCHL after atorvastatin treatment compared with control levels. After the bolus injection, chylo-apoB100 concentrations increased to a peak at 10 min in untreated FCHL patients (P < 0.05), and at 5 min in controls (P < 0.05) and were still elevated after 60 min in both groups compared with fasting levels. After atorvastatin, chylo-apoB100 did not change significantly. VLDL1 apoB100 increased only in untreated FCHL to a peak at 5 min (P < 0.01) and did not return to fasting levels, whereas no acute significant change was seen in treated FCHL patients or controls. In VLDL2, apoB100 levels did not change (Fig. 3Go).

Acute response of apoB48 and apoB100 after Intralipid (Fig. 4Go)

The acute response within 10 min of apoB48 in the chylo and VLDL2 fractions, calculated as total AUC, was significantly higher in untreated FCHL patients than in controls (Fig. 4Go, upper left panel), but the incremental AUCs did not reach statistical significance. Total and incremental AUCs did not decrease significantly after atorvastatin treatment in FCHL patients. In contrast, total and incremental AUCs for apoB100 decreased after atorvastatin treatment, reaching statistical significance for VLDL1 and also for VLDL2 (only incremental AUC). Overall, the effects of atorvastatin were more pronounced on the apoB100 fractions.



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  		FIG. 4. The mean (±SEM) acute response within 10 min in untreated FCHL ({blacksquare}), treated FCHL ({cjs2108}), and controls ({square}) of apoB48 and apoB100 calculated as AUC (A and C, respectively) and incremental AUCs (B and D, respectively) Significant changes are indicated: *, P < 0.05; {dagger}, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This report describes an acute increase in TRL-apoB in fasting FCHL subjects and healthy controls after a single injection of artificial TRLs (Intralipid), which does not contain apoB. Especially apoB100 and apoB48 in the chylomicron fraction (Sf, >400) increased after injection of artificial TRLs. The increases in apoB48 and apoB100 in large TRLs after injection of Intralipid in FCHL and controls represent, in our opinion, a marginated pool of these lipoproteins that are released into the circulation by competition with Intralipid particles for the same binding sites. The postinjection increase in cholesterol concentrations in the chylomicron fractions in untreated FCHL patients and controls also supports this conclusion. Another possible explanation for the acute increase in cholesterol is an increase in free circulating cholesterol absorbed from different tissues by the phospholipids in Intralipid. Alternatively, the postinjection cholesterol increase may have been caused by cholesterol enrichment of Intralipid particles mediated by cholesterol ester transfer protein (CETP) in both untreated FCHL patients and controls. This would also explain the lack of increase in cholesterol after Intralipid in treated FCHL patients, because atorvastatin is known to reduce CETP activity in combined hyperlipidemia (26). However, the (in vitro) lipid exchange by CETP is not a fast mechanism; therefore, it is not likely that this is the case. Although this cannot be fully excluded, such a CETP effect would not explain the acute rise in chylo-apoB48 and chylo-apoB100 after injection of Intralipid.

Findings similar to those described here have been reported previously. In an elegant study published by Hultin et al. (18), chylomicron metabolism was studied by radioactive labeled chylomicrons in rats. In that study, indications for margination were given, but the absolute concentration of apoB-containing particles was not determined, and the origins of the different circulating lipoproteins were not investigated. The margination of chylomicrons and VLDL1 has also been described in healthy subjects (19). The data presented in our paper are well in line with these results and provide evidence for margination of large TRLs, especially in situations where clearance is delayed, such as in FCHL. In addition, the present report extends those observations by showing a decreased margination of large TRLs by atorvastatin in FCHL subjects.

In contrast to the report by Karpe et al. (19), we measured apoB100 and apoB48 directly to differentiate VLDL from chylomicrons and their remnants. Our data suggest that margination might be a process lasting for many hours, because our participants had been fasting for 12 h and an acute postinjection increase was found for both apoB48 and apoB100. In our view, this represents detachment of these particles from binding sites to endothelial cells, because it is not likely that de novo production of these particles will occur within 10 min after injection of the emulsion.

Several explanations should be considered. Firstly, artificial TRLs may associate with apoE in vivo (27, 28) and may potentially compete with native TRLs for the same binding sites, thereby explaining the rise of apoB seen in large TRL fractions in the present study. Secondly, fusion of Intralipid particles with lipoproteins in higher densities (i.e. intermediate density lipoprotein or LDL) could result in a shift to a lower density, which could lead to higher concentrations in these latter fractions. This is not likely, because LDL apoB did not change after injection (data not shown). Finally, artificial TRLs may also associate with apoCII, as demonstrated previously (29). Competition with endogenous TRLs at the level of LPL may lead to accumulation of the latter by saturation of the common lipolytic pathway (30, 31). This may occur acutely, resulting in a rapid increase in apoB in TRL fractions as shown here. This latter explanation does not seem very likely, because atorvastatin treatment did not decrease the number of circulating fasting chylo-apoB100 and VLDL1 apoB100 in FCHL, and the postinjection increase that was observed before treatment was absent after treatment.

Margination of the TRLs seems to be a physiological process, because the healthy controls also showed an increase in apoB100-containing chylomicrons within 5 min, with less clear effects on chylo-apoB48.

In FCHL, remnant clearance is delayed, and VLDL particles are overproduced (6, 21, 22, 32, 33, 34). The larger extent of margination in FCHL could be explained by more circulating atherogenic lipoproteins caused by these two metabolic characteristics. It remains to be elucidated whether this exaggerated margination of TRLs also occurs in other situations characterized by overproduction and delayed clearance, such as in type 2 diabetes and obesity. Margination seems to be quantitatively more pronounced for apoB100-containing TRLs. ApoB48- and apoB100-containing remnants are removed by the same clearance mechanisms, the remnant receptor, hepatic lipase, LPL, and the LDL receptors, with a preference for apoB48-containing lipoproteins (30, 35). ApoB100-containing particles circulate longer and accumulate during the postprandial phase (6, 19), potentially leading to a larger marginated pool. Because humans are in a postprandial state most of the day (36), apoB100 accumulation is a daily phenomenon. The step-up titration was used to reinvestigate the patients receiving the most optimal dose of this statin to mimic the clinical situation. For this purpose, dose adjustment depended on fasting plasma TG and cholesterol levels. Unfortunately, fasting plasma TG could not be normalized by monotherapy with atorvastatin in all subjects. Cholesterol was significantly decreased in almost all fasting TRL fractions by atorvastatin treatment, which may have been caused by a decreased cholesterol synthesis in the liver (37). Most likely, decreased VLDL synthesis and up-regulation of the LDL receptors contributed to a decrease in plasma apoB by decreasing LDL apoB (38).

In different studies, a significant lowering of VLDL by atorvastatin was accomplished in coronary heart disease patients and in patients with combined hyperlipidemia by measuring the VLDL cholesterol levels (39, 40). In the present study, similar results were achieved. Another study recently published by Parhofer et al. (41) showed a significant decrease in small TRL apoB100 and small TRL apoB48 in 10 hypertriglyceridemic patients who were treated for 4 wk with atorvastatin (10 mg/d). These data are in contrast with those from the present study, probably due to the different groups of patients studied. Additionally, subfractionation of the TRLs was carried out differently, making a direct comparison inappropriate.

Statins increase hepatic LDL receptor expression and consequently lead to enhanced liver-mediated removal of apoB100-containing particles (42). Interestingly, there was no significant effect on fasting apoB48 in any of the TRL fractions, suggesting that the clearance of intestinally derived apoB-particles is influenced less by this statin in FCHL as has been reported recently by our group (34).

Atorvastatin also seems to have an effect on the marginated pool of TRLs according to the present data. The acute increase in chylo-apoB100 in untreated FCHL subjects and healthy controls and the increased VLDL-apoB100 in untreated FCHL were blunted after atorvastatin treatment. This could be explained in two different ways: atorvastatin may diminish the extent of margination, and/or atorvastatin may strengthen the binding of lipoproteins to the endothelial cells. As long as the margination process of TRLs is not clear, the second possibility cannot be excluded, albeit it is less likely because margination is a potentially harmful process, and statins have potent antiatherogenic effects, as observed in large intervention trials. In addition, if up-regulation of the endothelial LDL receptor by statins resulted in enhanced binding of TRLs to endothelial cells, lower fasting apoB48 and apoB100 concentrations in chylo and VLDL1 would have been expected after treatment.

A possible mechanism explaining margination as described here is LPL-mediated binding to endothelial cells (43). Furthermore, binding of LDL to the arterial wall by LPL has been described (44). Injection of Intralipid, increasing FFA, would then result in detachment, as reported by others (16, 17).

The dose of Intralipid used in this study was relatively low, and the effects observed may underestimate the total amount of marginated lipoproteins. Higher doses may lead to more pronounced effects. Additionally, earlier studies showed that Intralipid particles will only compete with lipoproteins that are bound to endothelial lipases and not with those bound to receptors (45).

What could the role of margination be in the development of atherosclerosis, and how could statins influence that? As suggested by our data, margination may already be present in the fasting state. Margination may be the first step in migration of apoB-containing lipoproteins through the vessel wall, even preceding subendothelial retention as suggested by others (20, 46). Another conclusion resulting from this paper is that margination is a physiological process, and it can be influenced by statin treatment. This margination is mainly caused by large TRLs, and because fasting concentrations of large apoB100-containing particles were not decreased by atorvastatin in FCHL, but the marginated pool was reduced, the effects of atorvastatin on these TRLs may be underestimated. The attachment of lipoproteins to the endothelial wall deserves more attention, and one of the putative mechanisms for binding of lipoproteins to the endothelium is via LPL or directly to proteoglycans (20). The exact mechanism by which atorvastatin reduces the marginated apoB pool remains to be elucidated.


   Footnotes
 
This work was supported by an unrestricted educational grant from Pfizer, Inc.

Abbreviations: apo, Apolipoprotein; AUC, area under the curve; BMI, body mass index; CETP, cholesterol ester transfer protein; FCHL, familial combined hyperlipidemia; FFA, free fatty acid; LPL, lipoprotein lipase; TG, triglyceride; TRL, triglyceride-rich lipoprotein.

Received December 17, 2003.

Accepted July 14, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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S. Lamon-Fava, M. R. Diffenderfer, P. H. R. Barrett, A. Buchsbaum, N. R. Matthan, A. H. Lichtenstein, G. G. Dolnikowski, K. Horvath, B. F. Asztalos, V. Zago, et al.
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