Published online before print July 27, 2006, doi: 10.1161/01.ATV.0000238348.05028.14
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© 2006 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Fenofibrate Reduces Atherogenesis in ApoE*3Leiden Mice
Evidence for Multiple Antiatherogenic Effects Besides Lowering Plasma Cholesterol
From Gaubius Laboratory (T.K., L.V., J.d.V.v.d.W., K.T., H.M.G.P., R.K.), TNO-Pharma, Leiden, The Netherlands; University of Ulm (W.K.), Department of Internal Medicine II-Cardiology, Ulm, Germany; Leiden University Medical Center (L.V., R.K.), Department of Vascular Surgery, Leiden, The Netherlands.
Correspondence to Robert Kleemann, PhD, Gaubius Laboratory, TNO-Pharma, P.O.Box 2215, 2301 CE Leiden, The Netherlands. E-mail R.Kleemann{at}pg.tno.nl or Robert.Kleemann@tno.nl
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Abstract |
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Objective— To demonstrate, quantify, and mechanistically dissect antiatherosclerotic effects of fenofibrate besides lowering plasma cholesterol per se.
Methods and Results— ApoE*3Leiden transgenic mice received either a high-cholesterol diet (HC) or HC containing fenofibrate (HC+FF) resulting in 52% plasma cholesterol-lowering. In a separate low-cholesterol diet (LC) control group, plasma cholesterol was adjusted to the level achieved in the HC+FF group. Low plasma cholesterol alone (assessed in LC) resulted in reduced atherosclerosis (lesion area, number and severity) and moderately decreased plasma serum amyloid-A (SAA) concentrations. Compared with LC, fenofibrate additively reduced lesion area, number and severity, and the total aortic plaque load. This additional effect in HC+FF was paralleled by an extra reduction of aortic inflammation (macrophage content; monocyte adhesion; intercellular adhesion molecule-1 [ICAM-1], soluble vascular cell adhesion molecule-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), MCP-1, and NF-
B expression), systemic inflammation (plasma SAA and fibrinogen levels), and by an upregulation of plasma apoE levels. Also, enhanced expression of ABC-A1 and SR-B1 in aortic macrophages may contribute to the antiatherosclerotic effect of fenofibrate by promoting cholesterol efflux.
Conclusion— Fenofibrate reduces atherosclerosis more than can be explained by lowering total plasma cholesterol per se. Impaired recruitment of monocytes/macrophages, reduced vascular and systemic inflammation, and stimulation of cholesterol efflux may all contribute to these beneficial effect of fenofibrate.
It is becoming increasingly clear that factors other than cholesterol determine atherogenesis. Using ApoE*3-Leiden mice, we demonstrate that fenofibrate, a PPAR
-agonist, reduces atherosclerosis beyond the effect attributable to lowering total plasma cholesterol per se via mechanisms that may involve reduced vascular and systemic inflammation and increased macrophage cholesterol efflux.
Key Words: atherosclerosis • fibrates • inflammation • reverse cholesterol transport • pleiotrop
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Introduction |
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Atherosclerosis is a complex multifactorial disease of the large arteries and the leading cause of morbidity and mortality in industrialized nations.1 Hypercholesterolemia is a well-established risk factor for the incidence of atherosclerosis and its pathologic complications. Current therapies for treatment of atherosclerosis are generally directed at lowering cholesterol levels. Statins have been shown to effectively lower circulating cholesterol levels and to reduce cardiovascular causes of death.1 Yet, many (>50%) patients still experience adverse coronary events, despite statin therapy.2 Also, the rate at which atherosclerosis progresses can vary considerably at any given plasma cholesterol level, and 20% of myocardial infarctions occur in individuals with normal plasma cholesterol levels.3,4 This suggests that factors other than cholesterol also contribute to the development of atherosclerosis.
Accumulating evidence points to inflammation as a driving force of atherogenesis,2,5 and chronically elevated levels of circulating inflammation markers are independently predictive of atherosclerotic risk and mortality in otherwise healthy patients.6–8 Furthermore, an inverse relationship exists between high-density lipoprotein (HDL) cholesterol and atherosclerosis in the general population,1,5,9 possibly related to the antioxidative and antiinflammatory properties of HDL as well as to its role in reverse cholesterol transport (RCT).9,10
Fibrates are potent lipid-lowering drugs to treat hypertriglyceridemia and mixed dyslipidemia.11 They efficiently lower plasma triglycerides and low-density lipoprotein (LDL) cholesterol, and increase HDL cholesterol by upregulating the hepatic gene expression and synthesis of apoAI, the major apolipoprotein of HDL.11 Fibrates exert their activity via activation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR ). PPAR , in conjunction with retinoid X receptor, positively regulates the transcription of target genes through binding to specific gene promoter response elements. This mode of action is particularly important for the regulation of genes that control lipid and lipoprotein metabolism and may, in large part, explain the normolipidemic action of fibrates.12 Independent of this mechanism, PPAR can also act as a negative regulator of pro-inflammatory genes via antagonizing the activity of inflammatory transcription factors.13–15 Together, these properties may result in an overall beneficial action of fibrates on cardiovascular disease.11,12 A large part of this effect of fibrates is independent of their effect on the plasma lipoprotein levels per se, because the changes in major lipids and lipoproteins could account for <25% of the beneficial effects of fibrate therapy.11,16 In line with this, pleiotropic effects of fibrates have been demonstrated: fibrates strongly reduced circulating markers of inflammation and suppressed cytokine-induced acute phase response reactions in mice and humans, independent of an effect on plasma cholesterol.15,17,18
Based on these findings and these characteristics of fibrates we hypothesized that fibrates may exert beneficial effects on atherosclerotic lesion development by mechanisms involving both lipid-lowering (ie, hypocholesterolemic) effects and effects not directly linked to this, eg, antiinflammatory and RCT-stimulating effects.
Previous studies directed at evaluating the role of fibrates in atherogenesis in mice, viz. in apoE-deficient and LDLR-deficient animals, were hampered by model-inherent drawbacks.19–21 Here, we used ApoE*3Leiden (E3L) transgenic mice, a well-established mouse atherosclerosis model. E3L mice display a lipoprotein profile comparable to that of patients with dysbetalipoproteinemia, ie, plasma cholesterol and triglyceride levels are mainly confined to the very-low-density lipoprotein (VLDL)/LDL fraction,22 and respond to hypolipidemic drugs in a similar way as humans.22,23 These characteristics, in combination with the possibility to titrate plasma cholesterol levels of E3L mice to a desired level by adjusting their dietary cholesterol intake, enabled us to quantify the fenofibrate-evoked reduction in atherosclerosis with particular emphasis on its antiinflammatory properties and its reverse cholesterol transport promoting activities.
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Methods |
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Animals
Female E3L transgenic mice (TNO-Pharma, Gaubius Laboratory, Leiden, The Netherlands) were characterized for expression of human apoE by enzyme-linked immunosorbent assay (ELISA). Mice were 12 weeks of age at the beginning of the study. Animal experiments were approved by the Institutional Animal Care and Use Committee of The Netherlands Organization for Applied Scientific Research (TNO) and were in compliance with European Community specifications.
Diets
During a run-in period of 3 weeks, 51 female E3L mice received an atherogenic, 0.50% w/w cholesterol containing Western-type diet24 further referred to as high-cholesterol (HC) diet. Then, mice were subdivided into 3 groups (n=17). In one group, HC diet feeding was continued for another 18 weeks (HC group). The fenofibrate-treated group (HC+FF group) received the same diet as the HC group but supplemented with fenofibrate (Sigma Aldrich). Based on the food intake in the HC+FF group, the daily dose of fenofibrate was 30 mg/kg bodyweight. The low-cholesterol control group (LC group) received the same Western-type diet24 but containing only 0.05% w/w cholesterol to reach a comparable plasma cholesterol level as the HC+FF group.
Analysis of Plasma Lipids, Lipoproteins, and Plasma Inflammation Markers
Total plasma cholesterol and triglyceride levels were measured after 4 hours of fasting, using kits No.1489437 (Roche Diagnostics, Almere, The Netherlands) and No.337-B (Sigma Diagnostics), respectively. For lipoprotein profiles, pooled plasma was fractionated using an ÅKTA fast protein liquid (FPLC) system (Pharmacia, Roosendaal, The Netherlands).23
For analysis of apolipoproteins, (fractionated) plasma samples were analyzed according to a described SDS-PAGE/Western blotting procedure13 using the following antibodies: anti-mouse apoE (Santa Cruz, Heerhugowaard, The Netherlands; sc-6384), anti-mouse apoAI- and apoB-specific antibodies (prepared at TNO-Pharma) and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz; sc-2304 and sc-2305).
The plasma levels of sVCAM-1 (R&D Systems), SAA (Biosource), and fibrinogen (in-house assay) were determined by ELISA as reported.22
Atherosclerotic Lesion Analysis
After 18 weeks of treatment, mice were euthanized to collect hearts and aortas. Hearts were fixed and embedded in paraffin to prepare serial cross sections (5-µm-thick) throughout the entire aortic valve area for (immuno)histological analysis. Cross-sections were stained with hematoxylin-phloxine-saffron (HPS), and atherosclerosis was analyzed blindly in 4 cross-sections of each specimen (at intervals of 30 µm) as described.23 QWin-software (Leica) was used for morphometric computer-assisted analysis of lesion number, lesion area, and lesion severity23 according to the classification of the American Heart Association.25 Analysis of atherosclerosis in longitudinally opened oil-red O-stained aortas has been described in detail elsewhere.23
Monocytes and macrophages were immunostained in cross-sections adjacent to the ones used for quantification of atherosclerosis using AIA31240 (1:3000; Accurate Chemical and Scientific, Brussels, Belgium) to determine the number of monocytes attached to the endothelium and the macrophage-containing lesion area.22,23 SMC area was determined as described.23 For immunostaining of ICAM-1, MCP-1, and NF-kB/p65, antibodies CBL1316 (Chemicon International, Heule, Belgium), sc-1784 and sc-8008 (Santa Cruz) were used, respectively.
Nucleic Acid Extraction and Gene Expression Analysis
Total RNA extraction was performed using RNAzol (Campro Scientific, Veenendaal, The Netherlands) and glass beads according to the manufacturer’s instructions. Then, cDNA was prepared using kit #A3500 (Promega, Leiden, The Netherlands) for real-time polymerase chain reaction (RT-PCR) analysis. The mastermix (Eurogentec, Seraing, Belgium), an ABI-7700 system (PE Biosystems, Nieuwekerk a/d Ijssel, The Netherlands) and established primer/probe sets18,23,26,27 were used according to the manufacturer’s instructions. Cyclophilin (PE Biosystems) was used as a reference.
Statistical Methods
Significance of differences was calculated by 1-way analysis of variance (ANOVA) test followed by a least significant difference (LSD) post hoc analysis using SPSS 11.5 for Windows (SPSS, Chicago, Ill). The level of statistical significance was set at P<0.05.
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Results |
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Fenofibrate Reduces Plasma Lipids in E3L Mice
The food intake was on average 2.6±0.1 grams per day in HC, which is comparable to that in HC+FF (2.7±0.1 g/d) and LC (2.8±0.1 g/d). There was no significant difference in body weight gain between the experimental groups.
Plasma cholesterol levels in HC remained at a constant level (on average 25.5±3.4 mmol/L; Figure 1A). Fenofibrate rapidly, within 1 week, reduced cholesterol to 8.6±2.1 mmol/L (P<0.01), a decrease of 66%. Cholesterol levels of HC+FF remained strongly reduced and were on average 12.4±2.8 mmol/L. The dietary cholesterol intake in LC was adjusted to match the plasma cholesterol levels achieved in HC+FF. With the exception of week 1 and 3, plasma cholesterol levels did not significantly differ between HC+FF and LC.
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Integrated over the whole treatment period, the total cholesterol exposure of HC was 517±42 mmol/L times weeks (Figure 1B). The total cholesterol exposure of HC+FF and LC did not significantly differ from each other and was 52% (P<0.01) less than that of HC.
Fasting plasma triglyceride levels in HC were on average 1.45±0.50 mmol/L and did not significantly change over time (Figure 1C). Fenofibrate-treatment reduced plasma triglyceride concentrations within 1 week to 0.25±0.06 mmol/L, a decrease of 83% (P<0.01); this low level was maintained during the remainder of the study. Triglyceride concentrations in LC averaged 1.2±0.4 mmol/L, which is comparable to HC and thus markedly higher than in HC+FF.
Fenofibrate Reduces Atherosclerosis Beyond the Effect Attributable to Lowering Total Plasma Cholesterol Per Se
After 18 weeks of experimental treatment, atherosclerosis was analyzed in cross-sections of the aortic valve area. The average lesion number per mouse was 10.2±3.3 in HC (Figure 2A). In HC+FF, the number of lesions was reduced by 89% (P<0.01), whereas the cholesterol-matched LC group showed only a 67% (P<0.01) reduction, a 22% (P<0.04) lesser decrease in number of lesions than in HC+FF.
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Measurement of the total cross-sectional lesion area revealed a similar additional effect of fenofibrate (Figure 2B). Whereas HC displayed a cross-sectional lesion area of 291 000±31 700 µm2, cholesterol-lowering alone (LC) reduced the lesion area by 87% (P<0.01) while with fenofibrate a further reduction was seen (89% versus LC; P<0.05).
The total plaque load in longitudinally opened, en face oil-red O-stained aortas of HC was 4.2±1.8 mm2 (not shown). LC displayed a 67% (P<0.01 versus HC) smaller plaque load, and fenofibrate further reduced this area by an additional 28% (P
0.05 versus LC). Together, these data indicate that fenofibrate reduces atherosclerosis development more than can be explained by lowering plasma cholesterol alone.
To assess differences in lesion severity, cross-sections of the aortic root were analyzed and lesions were graded25 (Figure 2C). In HC, no lesion-free sections were detectable; the analyzed cross-sections contained either mild type I-III (31% of all cross-sections) or severe type IV-V (69% of all cross-sections) atherosclerotic lesions. In LC, 63% of all cross-sections were lesion-free, 25% contained mild and 13% contained severe lesions. In HC+FF, 90% of all cross-sections were lesion-free and only 10% contained mild lesions; severe lesions were not observed.
Together, these data demonstrate that fenofibrate strongly reduces initiation and progression of atherosclerotic lesion formation and more than can be explained by lowering plasma cholesterol alone.
Fenofibrate Reduces Inflammatory Processes in the Arterial Wall
To evaluate whether the above described additional effects of HC+FF versus LC are paralleled by a reduced inflammatory state of the aorta, we evaluated various inflammation-related parameters, with particular emphasis on the contribution of macrophages.
In HC, the absolute macrophage-containing lesion area was 69 500±9400 µm2, which was reduced by 82% (P<0.01) in LC (P<0.01) and by 99% (P<0.01) in HC+FF (Figure 3A). The additional 17% reduction in HC+FF was significantly (P<0.01) different from LC.
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The macrophage density, ie, the macrophage area expressed as percentage of the total cross-sectional lesion area, did not significantly differ between HC (10±2%) and LC (10±3%), but fenofibrate strongly (by 86%) and significantly (P<0.01) lowered the macrophage density (Figure 3B).
To further explore the additional effects of fenofibrate, we analyzed whether fenofibrate inhibits the recruitment of macrophage precursor cells, blood monocytes. Whereas monocyte adhesion in LC was not significantly different from that in HC, HC+FF indeed showed a strongly and significantly reduced monocyte adhesion, equal to the level observed in chow-fed E3L mice (Figure 3C).
These cell-related effects were paralleled by a strongly diminished endothelial expression of the intercellular adhesion molecule ICAM-1 and a lower plasma level of sVCAM-1 in HC+FF (Figure 3D, 3E, and 3G). Furthermore, the total number of cells per cross-section with positive p65-NFB-staining (in cytosol and/or nucleus) was strongly reduced in HC+FF (Figure 3F). Assessment of p65-NFB–stained endothelial cells (ECs) per cross-section revealed a comparable number of p65-NFB-positive ECs in HC (17.9±5.6%) and LC (13.6±9.6%), but a strong reduction in HC+FF (0.8±1.2%; P<0.05 versus HC and LC). Also, the number of ECs showing active (ie, nucleus-associated) p65-NFB immunoreactivity in HC+FF (0.1±0.3%) was strongly and significantly (P<0.05) reduced compared with HC (2.3±5.6%) and LC (0.8±1.4%). These results indicate that fenofibrate not only reduces the number of p65-NFB-positive (endothelial) cells, but, most importantly, also the number of active p65-NFB containing ECs, thus providing a molecular explanation for the reduced monocyte adhesion in HC+FF.
The vascular expression of the pro-inflammatory chemokine MCP-1 and the monocyte/macrophage differentiation factor GM-CSF also was strongly reduced in HC+FF but not in LC (Figure 3H and 3I), suggesting that fenofibrate not merely impairs adhesion, but also recruitment and maturation of monocytes/macrophages.
Smooth muscle cell content, a measure of plaque stability, in LC was comparable to that in HC (3.84±2.52% and 4.45±0.5% of the total lesion area, respectively), but markedly reduced in HC+FF (1.60±1.05%, P<0.05 versus HC).
Fibrinogen and serum amyloid A (SAA) are 2 independent plasma inflammation markers which reflect the general systemic inflammatory state, and putatively participate in atherosclerotic lesion development. As shown in Figure 4, HC displayed higher plasma fibrinogen (3.3±0.5 mg/mL) and SAA (13.5±5.5 µg/mL) concentrations than chow-fed E3L mice (1.8±0.3 mg/mL fibrinogen; 3.2±2.0 µg/mL SAA). LC diet did not (fibrinogen) or only moderately (SAA) reduce the inflammation markers, but fenofibrate markedly lowered plasma fibrinogen and SAA levels by 33% (P<0.01) and 61% (P<0.01), respectively, relative to LC.
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Effect of Fenofibrate on Lipoprotein Profiles and Cholesterol Efflux
Clinical data have shown a strong inverse relationship between plasma levels of HDL and its major apolipoprotein, apoA1, and the incidence of atherosclerotic vascular disease.
Analysis of plasma apoA1 protein levels showed no significant differences between HC and LC, whereas a decrease of plasma apoA1 levels (58% reduction) and hepatic apoA1 mRNA expression levels (49% reduction) was found in HC+FF as determined by immunoblotting and RT-PCR, respectively (data not shown).
Figure 5A depicts the lipoprotein profiles for cholesterol after 12 weeks of experimental treatment. Grosso modo, the 3 experimental groups did not show significant differences in their HDL cholesterol levels, but in HC+FF and LC, the amount of very-low-density lipoprotein cholesterol and intermediate-density lipoprotein (IDL) cholesterol was strongly reduced relative to HC. Strikingly, in HC+FF a peak emerged in the transition area between small IDL/LDL particles and large HDL particles (fractions 10 to 17). To further define the HDL-related fractions and to characterize the fenofibrate-induced peak at fractions 10 to 16, lipoprotein fractions were analyzed by SDS-PAGE and Western blotting (Figure 5B). In all groups, fractions 
17 contained mainly the HDL-specific apoAI but only little amounts of apoE and no apoB. Fractions 12 to 16, corresponding to the fenofibrate-induced peak, were rich in apoB100 and apoE. Analysis of unfractionated plasma showed in HC+FF, but not in LC, significantly increased plasma apoE levels (Figure 5C), indicating that fenofibrate treatment is associated with elevated plasma apoE levels.
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A major mechanism by which apoA1 and HDL reduce atherosclerosis is by promoting cholesterol efflux from peripheral macrophages and returning it to the liver for excretion into the bile (RCT). Cholesterol efflux involves several gene products, including ATP binding cassette (ABC)-A1 cholesterol transporter and the scavenger receptor class B type I (SR-B1), each of which could be a target for stimulating this process. Aortic immunostaining revealed a predominantly macrophage-associated expression of both cholesterol transporters as demonstrated by a strong overlap with a staining specific for macrophages (supplementary information I, available online at http://atvb.ahajournals.org). RT-PCR analysis showed that aortic ABC-A1 and SR-B1 mRNA expression levels in HC+FF and LC were comparable, but reduced when compared with HC (Figure 5D, left). Taking into consideration that ABC-A1 and SR-B1 are mainly expressed in macrophages and that the macrophage content is diminished in HC+FF (compared with HC and LC; see also Figure 3A and 3B), ABC-A1 and SR-B1 mRNA levels were also determined relative to the mRNA concentration of a macrophage marker, CD14. Relative to CD14, the mRNA expression levels of ABC-A1 and SR-B1 were comparable in HC and LC, but a significant increase was observed in HC+FF (Figure 5D, right). These data point to an elevated expression of ABC-A1 and SR-B1 per macrophage with fenofibrate and are in agreement with published data.20,37
Also, in a separate experiment, peritoneal macrophages isolated from E3L mice treated with fenofibrate for 3 weeks displayed significantly increased SR-B1 mRNA expression levels when compared with placebo-treated E3L mice (not shown). Enhanced cholesterol efflux from the vasculature may thus contribute to fenofibrate’s pleiotropic, antiatherosclerotic effect.
For comparison, SR-B1 and ABC-A1 expression was also analyzed in livers. In LC, decreased mRNA expression levels of ABC-A1 and SR-B1 were found (33±4% and 62±13% of values in HC, respectively). In HC+FF, hepatic ABC-A1 mRNA expression remained high (99±7% of HC), but hepatic SR-B1 mRNA expression levels were strongly reduced (13±5% of HC), also in comparison with LC (supplementary information I).
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Discussion |
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In this report we evaluated the antiatherosclerotic effect of the hypolipidemic drug fenofibrate, a compound that not merely lowers cholesterol but reportedly also exhibits potential vasculoprotective effects besides cholesterol-lowering, including antiinflammatory effects. Comparison of fenofibrate-treated mice with a cholesterol-matched LC group allowed us for the first time to quantify and to comprehensively characterize pleiotropic antiatherosclerotic effects of a fibrate in vivo.
Most importantly, we demonstrate that the antiatherosclerotic effect of fenofibrate by far exceeds the effect that can be ascribed to its total plasma cholesterol-lowering activity per se. The effect of cholesterol-lowering alone on atherosclerosis has been assessed in the LC group, which displayed a markedly reduced aortic plaque load. This was accompanied by a moderate reduction of lesion number and severity and a relatively small decrease in plasma SAA concentrations. As compared with LC, the fenofibrate-treated HC+FF group displayed an additionally reduced aortic plaque load, lesion number, cross-sectional lesion area, and plaque severity. These additional effects in HC+FF were paralleled by a strong reduction of aortic inflammation (aortic macrophage content and density; monocyte adhesion; ICAM-1, MCP-1, GM-CSF, p65-NFB expression) and systemic inflammation (SAA, fibrinogen), and increased levels of factors important for cholesterol efflux from vascular macrophages (apoE; ABC-A1, SR-B1).
Data from recent clinical studies support the notion that activities of fibrates independent of normalizing plasma lipid levels may account for much of their cardioprotective effect.11 For example, the reduction of coronary heart disease-related mortality achieved with gemfibrozil in the VA-HIT prevention trial can be explained for only 
25% by changes in major lipid and lipoproteins, leaving most of the benefit of fibrate therapy as yet unexplained.16 The mechanisms responsible for this outcome are presently a matter of speculation, and the few animal studies that demonstrate fibrate effects unrelated to lipid-lowering are only suggestive in the context of cardiovascular disease.15,18 Our data unequivocally demonstrate and quantify for the first time to our knowledge that fibrates can reduce atherosclerosis beyond the effect attributable to their total plasma cholesterol-lowering effect per se and provide a molecular explanation for these additional beneficial effects.
Chronic subacute inflammation is a driving force of atherosclerotic lesion development5 and is reflected by elevated levels of systemic plasma inflammation markers, among which are CRP, SAA, and fibrinogen. These liver-derived markers have been demonstrated to independently predict future cardiovascular risk,8,28 and CRP reduction after cholesterol-lowering therapy is associated with improved clinical outcomes.29 Patients treated with fenofibrate at a dose comparable to the dose used in this study display reduced plasma levels of CRP, SAA, and fibrinogen after only 4 weeks of therapy.15 Here we demonstrate that fenofibrate reduces plasma SAA and fibrinogen levels in the HC+FF group beyond lowering plasma cholesterol per se (as assessed in cholesterol-matched LC group). This additional effect is in accordance with the downregulation of plasma CRP and fibrinogen levels by fenofibrate in absence of an effect on plasma cholesterol in human CRP transgenic mice by a mechanism involving quenching of the pro-inflammatory transcription factor NFB.18
In E3L mice, fenofibrate reduced plasma SAA and fibrinogen levels after already 3 weeks of treatment, ie, before atherosclerotic lesions have developed under the conditions applied.22,23
Because SAA and fibrinogen may participate in pro-atherogenic processes of early lesion evolution and promote lesion development,28,30 downregulation of their effective plasma concentrations by fenofibrate may possibly contribute to the observed pleiotropic antiatherosclerotic effects of the drug.
Our data provide evidence that fenofibrate exerts direct antiinflammatory effects in aorta: the macrophage density in atherosclerotic lesions was 86% smaller in HC+FF when compared with LC. This strong effect of fenofibrate on the aortic macrophage content may be explained by the observed suppression of endothelial monocyte adhesion in combination with the reduced aortic expression of ICAM-1 (which mediates the interaction between circulating monocytes and endothelial cells), MCP-1 (which stimulates the transmigration of monocytes through the endothelial cell layer), and GM-CSF (which augments the proliferation of vascular macrophages). In line with our data, a decrease of aortic MCP-1 mRNA expression was also observed in the descending aortas of fenofibrate-treated apoE-deficient mice.20 Downregulation of GM-CSF by a PPAR-activator has to our knowledge not been reported before. The molecular mechanism underlying these antiinflammatory vascular effects may well be the capacity of fenofibrate to suppress NF-B expression and activity, (Figure 3)13,14,18,31 a transcriptional master regulator that controls the expression of adhesion molecules, MCP-1, and GM-CSF.
Low HDL-cholesterol levels constitute a risk factor for CVD and are, independently of LDL-cholesterol levels, predictive for the risk of a cardiovascular event.32 Several clinical studies demonstrated that fibrate treatment results in a significant increase of HDL levels.11 This effect in humans can be explained by a PPAR-dependent upregulation of apolipoprotein apoA1.33 In rodent liver, apoA1 is negatively regulated by fibrates, including fenofibrate,33,34 a finding that was confirmed in this study.
Elevated plasma triglyceride levels represent an independent risk factor for cardiovascular disease. Fenofibrate treatment reduced plasma triglyceride concentrations by >80%. This result together with the changes observed in lipoprotein profile points to changes in the lipid content of lipoproteins and their size; this may modulate the atherogenicity of the lipoproteins. Triglyceride-rich lipoproteins even if smaller in the IDL fraction may be more atherogenic than triglyceride-poor IDL particles. Furthermore, these changes in physicochemical characteristics of lipoproteins may also affect their catabolism by tissues and cells, thus contributing differentially to atherosclerosis.
The cholesterol transporters ABC-A1 and SR-B1 are important for the efflux of macrophage-laden cholesterol from peripheral tissues and thereby for RCT.10,35,36 Our finding that the macrophage expression of ABC-A1 and SR-B1 in aorta is elevated by fenofibrate may be of relevance for the antiatherosclerotic activities of this compound, and is in line with several other studies showing that PPAR-activators stimulate the ABC-A1 pathway to induce cholesterol removal from human macrophage foam cells.20,37 Our in vivo effects of fenofibrate on ABC-A1 and SR-B1 as well as apoE expression may all contribute to enhanced aortic cholesterol efflux and thereby to reduced progression of atherosclerosis.
Taken together, we describe and quantify for the first time to our knowledge in a comprehensive way that the PPAR-agonist fenofibrate reduces atherosclerosis more than can be achieved by lowering total plasma cholesterol alone. We provide a mechanistic explanation for these additional beneficial effects by demonstrating diminished recruitment of monocytes/macrophages to atherosclerosis-prone sites of the aorta, reduced vascular inflammation, decreased expression of pro-atherogenic liver-derived acute phase reactants, and stimulation of factors involved in vascular cholesterol efflux. Our data underline the potential use of PPAR-agonists in the treatment of atherosclerosis and support the view that pharmaceutical intervention beyond standard LDL-lowering strategies may further reduce atherogenesis.
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Acknowledgments |
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We thank A. Jie for excellent technical assistance.
Sources of Funding
The Netherlands Organization for Scientific Research NWO and the Netherlands Heart Foundation NHS supported this work (NWO grant VENI 016.036.061 to R.K. and NHS grant 2002B102 to L.V. and K.T.). We thank Aventis Pharma, Frankfurt, Germany (support to H.P.) and the "New Initiative Systems Biology" research program of TNO (support to T.K. and R.K.) for supporting parts of this study. J. de Vries-van der Weij received grants support from the Center for Medical Systems Biology (CMSB) grant 115.
Disclosures
None.
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Footnotes |
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T.K. and L.V. contributed equally to this work.
Original received January 9, 2006; final version accepted July 18, 2006.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Braunwald E. Shattuck lecture—cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med. 1997; 337: 1360–1369.
[Free Full Text] - Libby P, Aikawa M. Stabilization of atherosclerotic plaques: new mechanisms and clinical targets. Nat Med. 2002; 8: 1257–1262.[CrossRef][Medline] [Order article via Infotrieve]
- Kannel WB. Range of serum cholesterol values in the population developing coronary artery disease. Am J Cardiol. 1995; 76: 69C–77C.[CrossRef][Medline] [Order article via Infotrieve]
- Genest J Jr, McNamara JR, Ordovas JM, Jenner JL, Silberman SR, Anderson KM, Wilson PW, Salem DN, Schaefer EJ. Lipoprotein cholesterol, apolipoprotein A-I and B and lipoprotein (a) abnormalities in men with premature coronary artery disease. J Am Coll Cardiol. 1992; 19: 792–802.[Abstract]
- Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med. 2002; 8: 1211–1217.[CrossRef][Medline] [Order article via Infotrieve]
- Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation. 2001; 104: 365–372.
[Free Full Text] - Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med. 1999; 340: 115–126.
[Free Full Text] - Ridker PM, Stampfer MJ, Rifai N. Novel risk factors for systemic atherosclerosis: a comparison of C-reactive protein, fibrinogen, homocysteine, lipoprotein(a), and standard cholesterol screening as predictors of peripheral arterial disease. JAMA. 2001; 285: 2481–2185.
[Abstract/Free Full Text] - Spieker LE, Ruschitzka F, Luscher TF, Noll G. HDL and inflammation in atherosclerosis. Curr Drug Targets Immune Endocr Metabol Disord. 2004; 4: 51–57.[CrossRef][Medline] [Order article via Infotrieve]
- Lewis GF, Rader DJ. New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ Res. 2005; 96: 1221–1232.
[Abstract/Free Full Text] - Despres JP, Lemieux I, Robins SJ. Role of fibric acid derivatives in the management of risk factors for coronary heart disease. Drugs. 2004; 64: 2177–2198.[CrossRef][Medline] [Order article via Infotrieve]
- Pineda TI, Gervois P, Staels B. Peroxisome proliferator-activated receptor alpha in metabolic disease, inflammation, atherosclerosis and aging. Curr Opin Lipidol. 1999; 10: 151–159.[CrossRef][Medline] [Order article via Infotrieve]
- Kleemann R, Gervois PP, Verschuren L, Staels B, Princen HM, Kooistra T. Fibrates down-regulate IL-1-stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-NFkappa B-C/EBP-beta complex formation. Blood. 2003; 101: 545–551.
[Abstract/Free Full Text] - Delerive P, Gervois P, Fruchart JC, Staels B. Induction of IkappaBalpha expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-alpha activators. J Biol Chem. 2000; 275: 36703–36707.
[Abstract/Free Full Text] - Gervois P, Kleemann R, Pilon A, Percevault F, Koenig W, Staels B, Kooistra T. Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate. J Biol Chem. 2004; 279: 16154–16160.
[Abstract/Free Full Text] - Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH, Linares E, Schaefer EJ, Schectman G, Wilt TJ, Wittes J. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol. Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med. 1999; 341: 410–418.
[Abstract/Free Full Text] - Tanne D, Benderly M, Goldbourt U, Boyko V, Brunner D, Graff E, Reicher-Reiss H, Shotan A, Mandelzweig L, Behar S. A prospective study of plasma fibrinogen levels and the risk of stroke among participants in the bezafibrate infarction prevention study. Am J Med. 2001; 111: 457–463.[CrossRef][Medline] [Order article via Infotrieve]
- Kleemann R, Verschuren L, De Rooij BJ, Lindeman J, De Maat MM, Szalai AJ, Princen HM, Kooistra T. Evidence for anti-inflammatory activity of statins and PPAR{alpha}-activators in human C-reactive protein transgenic mice in vivo and in cultured human hepatocytes in vitro. Blood. 2004; 103: 4188–4194.
[Abstract/Free Full Text] - Tailleux A, Torpier G, Mezdour H, Fruchart JC, Staels B, Fievet C. Murine models to investigate pharmacological compounds acting as ligands of PPARs in dyslipidemia and atherosclerosis. Trends Pharmacol Sci. 2003; 24: 530–534.[CrossRef][Medline] [Order article via Infotrieve]
- Duez H, Chao YS, Hernandez M, Torpier G, Poulain P, Mundt S, Mallat Z, Teissier E, Burton CA, Tedgui A, Fruchart JC, Fievet C, Wright SD, Staels B. Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice. J Biol Chem. 2002; 277: 48051–48057.
[Abstract/Free Full Text] - Fu T, Mukhopadhyay D, Davidson NO, Borensztajn J. The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions. J Biol Chem. 2004; 279: 28662–28669.
[Abstract/Free Full Text] - Kleemann R, Princen HM, Emeis JJ, Jukema JW, Fontijn RD, Horrevoets AJ, Kooistra T, Havekes LM. Rosuvastatin reduces atherosclerosis development beyond and independent of its plasma cholesterol-lowering effect in APOE*3-Leiden transgenic mice: evidence for antiinflammatory effects of rosuvastatin. Circulation. 2003; 108: 1368–1374.
[Abstract/Free Full Text] - Verschuren L, Kleemann R, Offerman EH, Szalai AJ, Emeis SJ, Princen HM, Kooistra T. Effect of low dose atorvastatin versus diet-induced cholesterol lowering on atherosclerotic lesion progression and inflammation in apolipoprotein E*3-Leiden transgenic mice. Arterioscler Thromb Vasc Biol. 2005; 25: 161–167.
[Abstract/Free Full Text] - van Vlijmen BJ, van den Maagdenberg AM, Gijbels MJ, Van Der BH, HogenEsch H, Frants RR, Hofker MH, Havekes LM. Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice. J Clin Invest. 1994; 93: 1403–1410.[Medline] [Order article via Infotrieve]
- Stary HC. Natural history and histological classification of atherosclerotic lesions: an update. Arterioscler Thromb Vasc Biol. 2000; 20: 1177–1178.
[Free Full Text] - Post SM, Groenendijk M, Solaas K, Rensen PC, Princen HM. Cholesterol 7alpha-hydroxylase deficiency in mice on an APOE*3-Leiden background impairs very-low-density lipoprotein production. Arterioscler Thromb Vasc Biol. 2004; 24: 768–774.
[Abstract/Free Full Text] - Verschuren L, Lindeman JH, Bockel JH, Abdul-Hussien H, Kooistra T, Kleemann R. Up-regulation and coexpression of MIF and matrix metalloproteinases in human abdominal aortic aneurysms. Antioxid Redox Signal. 2005; 7: 1195–1202.[CrossRef][Medline] [Order article via Infotrieve]
- Koenig W. Fibrin(ogen) in cardiovascular disease: an update. Thromb Haemost. 2003; 89: 601–609.[Medline] [Order article via Infotrieve]
- Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, McCabe CH, Pfeffer MA, Braunwald E. C-reactive protein levels and outcomes after statin therapy. N Engl J Med. 2005; 352: 20–28.
[Abstract/Free Full Text] - O’Brien KD, McDonald TO, Kunjathoor V, Eng K, Knopp EA, Lewis K, Lopez R, Kirk EA, Chait A, Wight TN, Debeer FC, Leboeuf RC. Serum amyloid A and lipoprotein retention in murine models of atherosclerosis. Arterioscler Thromb Vasc Biol. 2005; 25: 785–790.
[Abstract/Free Full Text] - Duval C, Chinetti G, Trottein F, Fruchart JC, Staels B. The role of PPARs in atherosclerosis. Trends Mol Med. 2002; 8: 422–430.[CrossRef][Medline] [Order article via Infotrieve]
- Assmann G, Schulte H. Relation of high-density lipoprotein cholesterol and triglycerides to incidence of atherosclerotic coronary artery disease (the PROCAM experience). Prospective Cardiovascular Munster study. Am J Cardiol. 1992; 70: 733–737.[CrossRef][Medline] [Order article via Infotrieve]
- Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation. 1998; 98: 2088–2093.
[Abstract/Free Full Text] - Berthou L, Duverger N, Emmanuel F, Langouet S, Auwerx J, Guillouzo A, Fruchart JC, Rubin E, Denefle P, Staels B, Branellec D. Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice. J Clin Invest. 1996; 97: 2408–2416.[Medline] [Order article via Infotrieve]
- Kennedy MA, Barrera GC, Nakamura K, Baldan A, Tarr P, Fishbein MC, Frank J, Francone OL, Edwards PA. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. Cell Metab. 2005; 1: 121–131.[CrossRef][Medline] [Order article via Infotrieve]
- Nakamura K, Kennedy MA, Baldan A, Bojanic DD, Lyons K, Edwards PA. Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein. J Biol Chem. 2004; 279: 45980–45989.
[Abstract/Free Full Text] - Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, Teissier E, Minnich A, Jaye M, Duverger N, Brewer HB, Fruchart JC, Clavey V, Staels B. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med. 2001; 7: 53–58.[CrossRef][Medline] [Order article via Infotrieve]
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