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Atherosclerosis 210 (2010) 381–387

Contents lists available at ScienceDirect

Atherosclerosis

journa l homepage: www.e lsev ier .com/ locate /a therosc leros is

itonavir protects against the development of atherosclerosis inPOE*3-Leiden mice

arion A.M. den Boera, Marit Westerterpa, Jitske de Vries-van der Weijb,d, Yanan Wanga, Lihui Hua,onia M.S. Espirito Santoa, Teake Kooistrad, Peter Reisse, Johannes A. Romijna,ouis M. Havekesa,c,d, Patrick C.N. Rensena,d,∗

Dept. of General Internal Medicine, Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, NetherlandsDept. of Human Genetics, Leiden University Medical Center, Leiden, NetherlandsDept. of Cardiology, Leiden University Medical Center, Leiden, NetherlandsNetherlands Organization for Applied Scientific Research-Quality of Life, Leiden, NetherlandsDept. of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam, Netherlands

r t i c l e i n f o

rticle history:eceived 18 March 2009eceived in revised form 5 November 2009ccepted 5 November 2009vailable online 1 December 2009

eywords:ardiovascular diseaseyslipidemiaigh-density lipoprotein

a b s t r a c t

Objective: The use of the HIV-protease inhibitor ritonavir (RTV) is associated with induction of hyper-triglyceridemia, which is a cardiovascular risk factor. Therefore, we investigated the effect of RTV onatherosclerosis development in APOE*3-Leiden transgenic mice, a model for human-like lipoproteinmetabolism and atherosclerosis.Methods and results: APOE*3-Leiden mice were fed a Western-type diet without or with RTV(35 mg/kg/day) for 19 weeks. RTV increased plasma TG levels throughout the study (∼2-fold; P < 0.05).Despite these increased TG levels, RTV decreased the atherosclerotic lesion area in the aortic root (−57%;P < 0.05), concomitant with reduced macrophage area (−72%; P < 0.01) and decreased lesion severity. Thiscould not be explained by reduced inflammatory markers in plasma (i.e. serum amyloid A, E-selectin and

rotease inhibitor fibrinogen), nor by decreased lipid accumulation in macrophages or increased cholesterol efflux frommacrophages, as assessed using peritoneal macrophages in vitro. Rather, whereas RTV did not affectplasma total cholesterol levels, RTV decreased (V)LDL-cholesterol and increased cholesterol in apoE-richlarge HDL.Conclusion: Despite inducing hypertriglyceridemia, RTV decreases atherosclerotic lesion area and sever-ity, associated with decreased (V)LDL-cholesterol and increased atheroprotective apoE-rich large HDL.

. Introduction

The introduction of highly active antiretroviral therapy (HAART)as considerably decreased morbidity and mortality associatedith HIV-infection. This therapy, however, is associated with a

ipodystrophy syndrome, which is characterized by changes in bodyat distribution and increased cardiovascular risk factors, such asyperlipidemia and insulin resistance [1,2].

The relationship between HAART and the development of pre-ature atherosclerosis in HIV-infected patients has not been

ully elucidated as yet. Studies measuring intima-media thicknessIMT) as a surrogate marker for the development of atheroscle-

∗ Corresponding author at: Leiden University Medical Center, Dept. of Endocrinol-gy and Metabolic Diseases, Room C4-R81, P.O. Box 9600, 2300 RC Leiden,etherlands. Tel.: +31 71 526 1830; fax: +31 71 524 8136.

E-mail address: P.C.N.Rensen@lumc.nl (P.C.N. Rensen).

021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2009.11.043

© 2009 Elsevier Ireland Ltd. All rights reserved.

rosis, do not conclusively show a correlation between HAARTand IMT [3–6]. Nonetheless, several studies observed a slightlyincreased risk for HIV-infected individuals treated with HAART forthe development of atherosclerosis [5,7–9]. The large prospectiveobservational study Data Collection on Adverse Events of Anti-HIVDrugs (DAD) showed that HAART was independently associatedwith an increased rate of myocardial infarction [10], which morerecently appeared to be confined to protease inhibitors [11].

Since it is difficult to study the effect of individual antiretrovi-ral drugs on the development of atherosclerosis in HIV-infectedsubjects, several mouse models have been used to this pur-pose. A study in male apoE knockout (apoE−/−) and LDL receptorknockout (LDLr−/−) mice showed that the HIV-protease inhibitor

ritonavir (RTV) promoted atherosclerotic lesion formation accom-panied by CD36-dependent cholesteryl ester (CE) accumulation inmacrophages [12]. In female LDLr−/− mice this effect was muchless pronounced [13], although female mice are in general moresusceptible to development of atherosclerosis than their male

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ounterparts [14]. This observation in transgenic mice only partlyupports the hypothesis that the metabolic effects of RTV mayltimately translate into an increased incidence of cardiovascularisease in HAART-treated subjects.

On the other hand, experimental drug studies in apoE−/− andDLr−/− mice not always predict human-like responses, as exem-lified by the inability of these mice to respond to the lipid-loweringffect of fibrates and statins [15]. This may be related to the crucialole of apoE in VLDL production and reverse cholesterol transport,nd to the role of the LDLr in the hepatic uptake of lipoprotein rem-ants. The APOE*3-Leiden transgenic mouse represents a modelith a more human-like lipoprotein metabolism. APOE*3-Leidenice have functional endogenous apoE and LDLr, and the APOE*3-

eiden transgene attenuates, rather than blocks, hepatic uptake ofemnants. As a consequence, the APOE*3-Leiden mouse representssuitable animal model to study the effects of dyslipidemia on

therosclerosis development [16], and responds in a human-likeanner to pharmacological intervention in lipid metabolism [15].Therefore, the aim of the present study was to determine the

ffect of RTV on the development of atherosclerosis in this APOE*3-eiden transgenic mouse model. We observed that RTV significantlyecreased atherosclerotic lesion area and severity in APOE*3-eiden mice, associated with decreased cholesterol in atherogenicV)LDL and increased cholesterol in apoE-rich large HDL.

. Methods

.1. Animals

Female APOE*3-Leiden transgenic mice, housed under standardonditions with free access to water and food, were used for exper-ments. Principles of laboratory animal care were followed and thenimal ethics committee of our institute approved all animal exper-ments.

.2. Diet-induced atherosclerosis development

Mice were fed a standard mouse chow diet (Hope Farms, Woer-en, Netherlands) until 8–10 weeks of age. After this period theyere fed a semi-synthetic Western-type diet (Hope Farms, Woer-en, Netherlands) containing 15% saturated fat, 0.2% cholesterolnd 40% sucrose for a 5 weeks run-in period. Mice were random-zed based on plasma TG levels and divided into two groups (n = 14ach). One group of APOE*3-Leiden mice was fed the Western-ype diet containing RTV (Norvir, Abbott, Kent, United Kingdom)orresponding to a dose of 35 mg/kg body weight/day (based onhe average daily food intake) for 19 weeks. The other group wased the Western-type diet without RTV to serve as an appropriateontrol. On the basis of two papers investigating pharmacokineticroperties of HIV-protease inhibitors in mice [17,18], we previ-usly designed a dose-finding study in which we observed thatTV at the dose of 35 mg/kg body weight/day induced hypertriglyc-ridemia without causing liver damage as reflected by non-affectedlasma levels of ALAT [19]. Taking into account that mice have a0-fold higher metabolism of xenobiotics than humans, this doseorresponds well to a human equivalent of 100 mg bid that inducesypertriglyceridemia in healthy volunteers [20].

.3. Analysis of plasma lipids and markers of inflammation

Every 4 weeks tail vein blood was collected into chilled

araoxon-coated capillary tubes to prevent in vitro lipolysis [21].hese tubes were placed on ice and immediately centrifuged at 4 ◦C.lasma levels of TG and total cholesterol were determined enzy-atically using commercially available kits and standards (#310-A

igma GPO-Trinder kit, St. Louis, MA, USA; CHOL MPR3, Boehringer,

erosis 210 (2010) 381–387

Mannheim, Germany). Plasma serum amyloid A (SAA), fibrinogenand E-selectin were determined after 16 weeks of RTV administra-tion using ELISAs as described previously [22].

2.4. Atherosclerosis analysis

After 19 weeks on the Western-type diet with or withoutRTV, mice were sacrificed. The hearts were perfused with ice-cold PBS, isolated, fixed in phosphate-buffered 4% formaldehyde,dehydrated and embedded in paraffin. The embedded hearts werecross-sectioned (5 �m) throughout the entire aortic root area. Sec-tions were stained with hematoxylin–phloxine–saffron (HPS). Permouse, four sections at 40 �m intervals were used for quantifica-tion of atherosclerotic lesion area and characterization of lesionseverity. Lesion area was determined using Image-Pro Plus version3.0 analysis software (Media Cybernetics, U.S.). The atheroscleroticlesions were categorized for severity according to the AmericanHeart System for humans [23], which has been adapted to cate-gorize lesions in mice [24]. Three categories were discerned: noor early lesions (type 0–1 lesions), moderate lesions that repre-sent fatty streaks containing only foam cells (type 2–3 lesions)or advanced lesions showing foam cells in the media and pres-ence of fibrosis, cholesterol clefts, mineralization and/or necrosis(type 4–5 lesions). The number observed in each lesion cate-gory is expressed as a percentage of the total number of lesionspresent within one group of mice. AIA 31240 antiserum (1:3000,Accurate Chemical and Scientific, Westbury, NY) was used toquantify macrophage area, and apoptotic cells were quantified byTdT-mediated dUTP Nick-End Labeling (TUNEL) staining with theDeadEnd Colorimetric TUNEL system (Promega Benelux, Leiden,The Netherlands).

2.5. In vitro lipid association and cholesterol efflux studies withperitoneal macrophages

Four days after intraperitoneal injection of Brewer’s thiogly-collate into chow-fed APOE*3-Leiden transgenic mice, peritonealcells were harvested into ice-cold PBS. The cells were pelleted bycentrifugation, and resuspended in DMEM (Invitrogen) containing10% fetal calf serum (Cambrex) and 1% penicillin and streptomycin.The cells were plated into 24-wells plates (Costar, Corning Inc.,Corning, NY, USA) at a density of 6.0 × 105 cells per well. Afterincubation at 37 ◦C under 5% CO2 humidified air for 2 h, cells werewashed to remove non-adhering cells. After o/n culturing, themacrophages were pre-incubated with RTV (0, 0.1 and 1 �g/mL)or vehicle (0.5% ethanol) for 24 h. To determine the effect of RTVon the association of oxLDL with the macrophages, cells weresubsequently incubated with 50 �g/mL oxLDL [25] and 2 �Ci/mL[3H]cholesterol (Amersham Biosciences, UK) in presence of RTVor vehicle for another 24 h. Alternatively, to determine the effectof RTV on the association of 80 nm-sized TG-rich VLDL-like emul-sion particles [26], cells were incubated with 380 �g TG/mL glyceroltri[3H]oleate (triolein, TO) and [14C]cholesteryl oleate (CO) double-labeled VLDL-like emulsion particles for 3 h. After incubation witheither oxLDL or TG-rich particles, cells were washed three timeswith ice-cold PBS, lysed with 0.1 M NaOH and the amount of cell-associated radioactivity was determined. To determine the effectof RTV on cholesterol efflux, cells were incubated with 50 �g/mLacLDL and 2 �Ci/mL [3H]cholesterol for 32 h. Subsequently, cellswere incubated with RTV (0, 0.1 and 1 �g/mL) for 16 h, followed byeither mRNA isolation for analysis of Abca1, Abcg1 and Sr-b1 gene

expression, or incubation of cells with human apoAI (10 �g/mL) orhuman HDL (50 �g/mL) in continued presence of RTV or vehiclefor 4 h. After the incubations, radioactivity was determined in themedium and in cell lysates to determine the percentage of choles-terol efflux.

erosclerosis 210 (2010) 381–387 383

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.6. Plasma lipoprotein profiling by FPLC

To determine the lipid and protein distribution over plasmaipoproteins, lipoproteins were separated using FPLC. Plasma wasooled per group and 50 �L of each pool was injected onto auperose 6 PC 3.2/30 column (Äkta System, Amersham Pharma-ia Biotech, Piscataway, NJ, USA) and eluted at a constant flow ratef 50 �L/min in PBS, 1 mM EDTA, pH 7.4. Fractions of 50 �L wereollected and assayed for cholesterol as described above. Equalmounts of every second fraction were diluted in sample buffernd subjected to SDS-PAGE (4–20% protein gels, BioRad). Proteinands were stained with Coomassie Brilliant Blue R250 (Sigma) andpparent molecular masses were identified.

.7. Statistical analysis

Differences between experimental groups were determinedy the Mann–Whitney U test for two independent samples. Theifferences in lesion severity were statistically tested using the Chi-quare test. The level of statistical significance of the differencesas set at P < 0.05. Analyses were performed using SPSS 12.0 forindows software (SPSS, Chicago, IL, USA).

. Results

.1. Ritonavir increases plasma TG

To evaluate the effect of RTV on atherosclerosis development,POE*3-Leiden mice were fed a Western-type diet without orith RTV added (35 mg/kg/day). At baseline and every 4 weeks

hereafter, blood samples were taken to determine plasma levelsf TG and total cholesterol (Fig. 1). Throughout the whole studyeriod, RTV administration significantly increased plasma TG levelspproximately 2-fold compared to control mice and this effect sus-ained until the end of the experiment (P < 0.05 for all time points;ig. 1A). In contrast, RTV did not affect plasma total cholesterolevels throughout the study period (Fig. 1B).

.2. Ritonavir decreases atherosclerotic lesion development

After 19 weeks on the Western-type diet without or withTV, mice were sacrificed to quantify the atherosclerotic lesionrea and to determine atherosclerotic lesion severity in the aorticoot (Fig. 2). RTV attenuated the development of atherosclero-is, as shown by representative pictures (Fig. 2A). Analysis ofhe lesions in all mice showed that RTV decreased the meantherosclerotic lesion area by 57% as compared to control mice22 ± 8 × 103 �m2 vs. 51 ± 9 × 103 �m2; P < 0.05; Fig. 2B). This waseflected by a reduction in moderate (type 2–3) and advancedtype 4–5) lesions, concomitant with an increase in the percent-ge of segments with early (type 1) lesions or no lesions at all (type) (P < 0.05; Fig. 2C). Further analysis of the lesions showed thatTV decreased the macrophage content by 72% (5 ± 2 × 103 �m2

s. 18 ± 3 × 103 �m2; P < 0.01; Fig. 2D), without significantly affect-ng the relative macrophage area as compared to the total lesionrea. In addition, RTV tended to increase the apoptotic cell numberpproximately 2-fold (P > 0.05; Fig. 2E).

.3. Ritonavir does not affect inflammatory parameters such asAA, fibrinogen and E-selectin

Since it was surprising that the hypertriglyceridemic effectf RTV did not translate into increased atherosclerosis, we setut to elucidate the potential mechanism(s) underlying thebserved reduction in atherosclerosis. As (systemic) inflamma-ion is causally involved in atherosclerosis, we first determined

Fig. 1. RTV increases plasma TG. Mice were fed a Western-type diet without or withRTV added (35 mg/kg bodyweight/day). At baseline and every 4 weeks thereafter,plasma levels of triglycerides (A) and total cholesterol (B) were measured after 4 hfasting. Values are means ± SD (n = 14 per group; *P < 0.05).

SAA, E-selectin and fibrinogen levels in plasma as a mea-sure of whole-body state of inflammation. However, after 16weeks of intervention, we found no differences with respect toplasma levels of SAA (28.3 ± 16.0 �g/mL vs. 37.1 ± 13.7 �g/mL),E-selectin (145 ± 17 �g/mL vs. 164 ± 35 �g/mL) and fibrinogen(1.7 ± 0.3 mg/mL vs. 2.1 ± 0.6 mg/mL) between the control groupand the RTV-treated group, respectively.

3.4. Ritonavir increases the association of lipids withmacrophages

It has been shown that RTV promotes atherosclerosis in apoE−/−

and LDLr−/− mice, as explained by increased CD36-dependent CEaccumulation in macrophages [12]. To evaluate whether RTV mayreduce, rather than increase, cholesterol uptake in macrophagesof APOE*3-Leiden mice, we investigated the effect of RTV onthe uptake of [3H]cholesterol-oxLDL by peritoneal macrophages.However, in line with the observations in macrophages fromapoE−/− and LDLr−/− mice, RTV dose-dependently enhanced thecell-association of oxLDL up to 28% at 1 �g/mL RTV (P < 0.05;

Fig. 3A). We have previously observed that RTV treatment ofAPOE*3-Leiden mice reduces the systemic expression of LPL, asreflected by reduced postheparin LPL levels [19]. Since a reduc-tion in LPL activity in macrophages could result in reduction ofTG-derived fatty acid uptake, we evaluated the effect of RTV on

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opment in the aortic root in APOE*3-Leiden mice, as reflected

ig. 2. RTV decreases atherosclerotic lesion area and severity. After 19 weeks on trea as well as lesion severity was determined in the aortic root. Representative oveotal lesion area was quantified (B), the severity of lesions was determined (C), theetermined (E). Values are means ± SEM (n = 12–14 per group; *P < 0.05; **P < 0.01).

he uptake of [3H]TO and [14C]CO double-labeled TG-rich VLDL-likemulsion particles by peritoneal macrophages. However, RTV dose-ependently increased the association of radioactivity derived fromoth [3H]TO (Fig. 3B) and [14C]CO (Fig. 3C) (P < 0.05). Since theatio between the uptake of [3H]TG and [14C]CO was similar forll conditions, and was equal to their ratio in the emulsion itself,e conclude that RTV increases whole-particle association rather

han selectively inducing the uptake of TG-derived fatty acids.To evaluate whether RTV may increase cholesterol efflux

rom cholesterol-laden macrophages of APOE*3-Leiden mice, wenvestigated the effect of RTV on the mRNA expression of theholesterol transporters Abca1, Abcg1 and Sr-b1 as well as the effluxf [3H]cholesterol from acLDL-laden peritoneal macrophages.owever, RTV did not increase the mRNA expression of these trans-orters (not shown) or the cholesterol efflux to apoAI (Fig. 3D) orDL (Fig. 3E).

.5. Ritonavir improves the lipoprotein profile by reducingholesterol in VLDL while increasing cholesterol in apoE-rich HDL

RTV did not affect plasma total cholesterol levels (Fig. 1A). How-ver, since the ratio of pro-atherogenic (V)LDL and anti-atherogenic

estern-type diet without or with addition of RTV, mice were sacrificed and lesions of the aortic root area of a control and of a RTV-treated mouse are shown (A). Thephage area of the lesions was quantified (D) and the number of apoptotic cells was

HDL is more important for atherosclerosis development than theplasma total cholesterol level, we determined the abundanceof the various lipoproteins in pooled plasma after separa-tion by FPLC. Indeed, RTV markedly affected the lipoproteinprofile (Fig. 4A). In addition to reducing (V)LDL-C by approx-imately 40%, RTV induced the formation of large apoE-richHDL-1, as evidenced by apolipoprotein detection after SDS-PAGE(Fig. 4B).

4. Discussion

The introduction of antiretroviral drug therapy has consider-ably increased the life span of HIV-infected patients. Consequently,concurrent long-term side effects become more clinically relevantin the considerations for the most optimal drug regimens. In thecurrent study we show that RTV decreases atherosclerosis devel-

by smaller atherosclerotic lesion area and the presence of lessmacrophages in the lesions, despite the induction of hypertriglyc-eridemia. Mechanistic studies showed that the anti-atherogeniceffect of RTV is most likely related to decreased (V)LDL-cholesteroland increased atheroprotective large apoE-rich HDL.

M.A.M. den Boer et al. / Atherosclerosis 210 (2010) 381–387 385

Fig. 3. RTV increases the association of oxLDL and TG-rich VLDL-like emulsion particles with peritoneal macrophages. Cells were pre-incubated for 24 h with RTV (0, 0.1 and1 �g/mL). Subsequently, cells were incubated with oxLDL (50 �g/mL) and [3H]cholesterol (2 �Ci/mL) in presence of RTV or vehicle for 24 h (A), or with glycerol tri[3H]oleate(TO) and [14C]cholesteryl oleate (CO) double-labeled VLDL-like particles (380 �g TG/mL) for 3 h (B and C). After the incubations, cells were washed, and the amount of cell-associated radioactivity was determined. Alternatively, cells were first incubated with aincubated with RTV (0, 0.1 and 1 �g/mL) for 16 h, followed by incubation of cells with huvehicle for 4 h. After the incubations, radioactivity was determined in the medium and in c(n = 3–4; *P < 0.05).

Fig. 4. RTV decreases VLDL-cholesterol and increases apoE-rich HDL-cholesterol.After 16 weeks on the Western-type diet without or with the addition of RTV, bloodwas drawn and plasma was pooled per treatment group (n = 14). Pooled plasmawas fractionated using FPLC, and the individual fractions were assayed for totalcholesterol (A) and the apolipoproteins apoAI and apoE (B).

cLDL (50 �g/mL) and [3H]cholesterol (2 �Ci/mL) for 32 h. Subsequently, cells wereman apoAI (10 �g/mL) or human HDL (50 �g/mL) in continued presence of RTV orell lysates to determine the percentage of cholesterol efflux. Values are means ± SD

We observed that RTV induces an increase in plasma TG (∼2-fold), as compared to control treatment, that persists throughoutthe whole study period of 19 weeks. This finding is consistentwith studies showing that administration of RTV to healthy humanvolunteers increases plasma TG by +27% at 100 mg bid [20] and+145% at 300–500 mg bid [27]. We have previously shown that thisincrease in plasma TG is specific for VLDL, and could be explainedby attenuation of the clearance of VLDL by a reduction in plasmaLPL activity [19]. Since TG are associated with an increased risk forthe development of cardiovascular disease [28], we expected thatthis persistent hypertriglyceridemic effect of RTV would be accom-panied by increased atherosclerosis development. In contrast, weobserved that RTV markedly reduced atherosclerosis lesion area, aswell as lesion severity.

It is tempting to speculate about the reason why RTV is anti-atherogenic in APOE*3-Leiden mice. Previous studies in maleapoE−/− and LDLr−/− mice showed that HIV-protease inhibitorsincluding RTV promoted atherosclerotic lesion formation inde-pendently of dyslipidemia [12]. Also in female LDLr−/− mice,RTV increased atherosclerosis, albeit to a lesser extent [13].These observations were explained by increased CD36 expressionin macrophages, thereby enhancing CD36-dependent cholesterylester accumulation [12]. On the other hand, antiretroviral therapy

markedly decreased CD36 expression in monocytes from healthyvolunteers, treatment-naive HIV-infected subjects, as well as inthe human monocyte cell line THP1 [29]. Therefore, we speculatedthat the different outcome in atherosclerosis between apoE−/− andLDLr−/− on one hand and APOE*3-Leiden mice on the other hand

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ay relate to divergent effects on CD36 expression. However, wehowed that RTV induced a dose-dependent increase in the CD36-ependent association of oxLDL with peritoneal macrophages fromPOE*3-Leiden mice. Although these data are in full accordanceith the previous macrophage studies [12], the apparent increase

n CD36 can thus not explain the decreased atherosclerosis inducedy RTV as observed in the current study. In fact, we observed thatTV showed a non-significant trend to reduce the macrophage area

n the lesion to a higher extent (72%) than the total lesion area (57%).It would also be conceivable that RTV increases cholesterol

fflux from macrophages, thereby reducing the macrophage arean the lesion. RTV has been shown to increase peroxisomeroliferator-activated receptor-� (PPAR�) [12], which could bexpected to reduce atherosclerosis by promoting cholesterol effluxia upregulation of ATP-binding cassette A1 (ABCA1) and ABCG130–33]. However, RTV did not upregulate the expression of Abca1,bcg1 and Sr-b1 by peritoneal macrophages and did not increaseholesterol efflux to either apoAI or HDL, which renders a poten-ial anti-atherosclerotic effect or RTV through PPAR� activationnlikely.

Another possible mechanism underlying the observed decreasen atherosclerosis could relate to effects of RTV on LPL activity. In ourrevious study, we observed that RTV considerably decreased totalPL activity in postheparin plasma (i.e. 50%) [19]. Since it has beenemonstrated by bone marrow transplantation that deletion ofPL from macrophages reduces atherosclerosis [34], we speculatedhat RTV may reduce LPL expression in macrophages. Interest-ngly, when we investigated this hypothesis in vitro, we foundhat RTV dose-dependently increased the association of both VLDL-ike emulsion-derived TG and cholesteryl esters with macrophages.hese findings indicate that RTV increases whole-particle uptake ofLDL-like emulsion particles by macrophages rather than inducingdecrease in LPL activity in these cells.

Protease inhibitors including RTV have been shown to induce ERtress and apoptosis in vitro [35] and in vivo [36]. Since RTV-inducedpoptosis may theoretically result in smaller lesions, apoptoticell number in the lesions was quantified by TUNEL staining. RTVended to increase the apoptotic cell number by 2-fold, whichid not reach statistical significance due to considerable inter-

ndividual variation. However, since apoptosis was measured onlyt the end point of the study, we cannot rule out that RTV may haveugmented apoptosis rate during lesion development, ultimatelyesulting in smaller lesions.

The RTV-induced reduction of atherosclerosis in APOE*3-Leidenice was associated with a pronounced decreased (V)LDL-

holesterol and increased large apoE-rich HDL. The reduction inV)LDL-cholesterol may well explain the reduction in atheroscle-osis, since numerous studies have shown that diet-inducedtherosclerosis development in APOE*3-Leiden mice is highlyependent on the plasma level of (V)LDL-cholesterol [15]. The RTV-

nduced decrease in (V)LDL-cholesterol is somewhat surprising,ince we previously observed that short-term RTV treatment forweeks resulted in a small increase in VLDL-cholesterol. Appar-

ntly, long-term RTV treatment results in an adaptation of theipoprotein profile, albeit that the underlying mechanism is stillnclear. In addition to reduced (V)LDL-cholesterol, the increase

n large apoE-rich HDL may be an additional explanation forhe observed reduction in atherosclerosis. HDL is regarded anti-therogenic by its role in reverse cholesterol transport and byts anti-inflammatory, anti-oxidative and anti-thrombotic proper-ies. Although it is unknown whether apoE-rich HDL is functional

ith respect to these various anti-atherogenic properties, accu-ulation of this particle in plasma of APOE*3-Leiden mice

pon treatment with e.g. fenofibrate [37] and the LXR agonist0901317 [38] is consistently associated with reduced atheroscle-osis.

[

erosis 210 (2010) 381–387

It is interesting to note that the effects of RTV on the choles-terol distribution in APOE*3-Leiden mice (i.e. decreased cholesterolwithin VLDL and increased cholesterol within large apoE-rich HDL)do not reflect the effects of RTV in healthy human volunteers (i.e.increased VLDL-cholesterol and decreased HDL-cholesterol) [27].This seeming discrepancy is most likely caused by the fact thatmice naturally lack expression of the cholesteryl ester transferprotein (CETP), which plays an important role in human choles-terol metabolism by mediating the transfer of cholesterol fromatheroprotective HDL particles to atherogenic (V)LDL particles inplasma. Previous studies have shown that large apoE-rich HDL thataccumulates in mice due to e.g. LCAT overexpression [39] or treat-ment with fenofibrate [40] is prevented by transgenic expressionof human CETP. Taken together with the fact that hypertriglyc-eridemia, as induced by RTV, is a driving force for CETP activity[41], it is thus likely that the presence of CETP is crucial for deter-mining the effect of RTV on the plasma cholesterol profile andpossibly also atherosclerosis. Therefore, CETP is likely to contributeto the adverse effect of RTV on the cholesterol distribution in humanplasma. Future studies addressing the contribution of CETP to theeffect of RTV on atherosclerosis development are thus warranted.

5. Conclusions

RTV decreases the development of atherosclerosis in APOE*3-Leiden transgenic mice, despite the induction of hypertriglyc-eridemia, which is most likely explained by decreased (V)LDL-cholesterol and increased atheroprotective apoE-rich large HDL.

Conflict of interest

None.

Acknowledgements

Sources of funding: This work was supported by the Leiden Uni-versity Medical Center (Gisela Thier Fellowship to Dr. Rensen), theNetherlands Organization for Scientific Research (NWO VIDI grant917.36.351 to Dr. Rensen; NWO grant 903-39-291 to Dr. Havekes),and the Centre for Medical Systems Biology (CMSB project 115 toDr. Havekes).

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