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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2560-2566
Published online before print August 24, 2006, doi: 10.1161/01.ATV.0000242904.34700.66
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2560.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Dual PPAR{alpha}/{gamma} Agonist Tesaglitazar Reduces Atherosclerosis in Insulin-Resistant and Hypercholesterolemic ApoE*3Leiden Mice

A. Susanne M. Zadelaar; Lianne S.M. Boesten; J. Wouter Jukema; Bart J.M. van Vlijmen; Teake Kooistra; Jef J. Emeis; Erik Lundholm; German Camejo; Louis M. Havekes

From the Department of Cardiology (A.S.M.Z., J.W.J., L.M.H.), Leiden University Medical Center; TNO-Gaubius Laboratory (A.S.M.Z., L.S.M.B., T.K., J.J.E., L.M.H.), Biomedical Research Unit; Department of General Internal Medicine (L.S.M.B., L.M.H.), Leiden University Medical Center; Hemostasis and Thrombosis Research Center (B.J.M.vV.), Leiden University Medical Center, Leiden, The Netherlands; AstraZeneca (E.L., G.C.), Mölndal, Sweden.

Correspondence to Louis M. Havekes, TNO-Gaubius Laboratory, Biomedical Research Unit, Zernikedreef 9, 2333 CK, P. O. Box 2215, 2301 CE Leiden, The Netherlands. E-mail LM.Havekes{at}pg.tno.nl


*    Abstract
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 *Abstract
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Objective— We investigated whether the dual PPAR{alpha}/{gamma} agonist tesaglitazar has anti-atherogenic effects in ApoE*3Leiden mice with reduced insulin sensitivity.

Methods and Results— ApoE*3Leiden transgenic mice were fed a high-fat (HF) insulin-resistance–inducing diet. One group received a high-cholesterol (HC) supplement (1% wt/wt; HC group). A second group received the same HC supplement along with tesaglitazar (T) 0.5 µmol/kg diet (T group). A third (control) group received a low-cholesterol (LC) supplement (0.1% wt/wt; LC group). Tesaglitazar decreased plasma cholesterol by 20% compared with the HC group; cholesterol levels were similar in the T and LC groups. Compared with the HC group, tesaglitazar caused a 92% reduction in atherosclerosis, whereas a 56% reduction was seen in the cholesterol-matched LC group. Furthermore, tesaglitazar treatment significantly reduced lesion number beyond that expected from cholesterol lowering and induced a shift to less severe lesions. Concomitantly, tesaglitazar reduced macrophage-rich and collagen areas. In addition, tesaglitazar reduced inflammatory markers, including plasma SAA levels, the number of adhering monocytes, and nuclear factor {kappa}B-activity in the vessel wall.

Conclusions— Tesaglitazar has anti-atherosclerotic effects in the mouse model that go beyond plasma cholesterol lowering, possibly caused by a combination of altered lipoprotein profiles and anti-inflammatory vascular effects.

We investigated whether the dual PPAR{alpha}/{gamma} agonist tesaglitazar has anti-atherogenic effects in ApoE*3Leiden transgenic mice with reduced insulin sensitivity. Tesaglitazar has anti-atherosclerotic effects that go beyond plasma cholesterol-lowering. This may be caused by a combination of altered lipoprotein profiles and anti-inflammatory vascular effects.


Key Words: atherosclerosis • cholesterol • inflammation • inhibitors


*    Introduction
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Agonists of the peroxisome proliferator-activated receptor (PPAR){alpha} have positive effects on lipid metabolism both in animal models and in clinical practice.1–3 Agonists of PPAR{gamma}, the thiazolidinediones rosiglitazone and pioglitazone, improve insulin resistance in type 2 diabetes, and pioglitazone improves the dyslipidemia associated with insulin resistance.4–6 In addition to these effects, both PPAR{alpha} and PPAR{gamma} agonists have anti-inflammatory properties7,8 that can provide additional cardiovascular benefit.9

PPAR{alpha} and PPAR{gamma} agonists could reduce atherosclerosis by improving the dyslipidemia of insulin resistance and by modulating the low-level chronic inflammatory response induced by this disease. Systemically, they ameliorate the atherogenic lipid profile by reducing plasma free fatty acids and triglycerides, and increasing high-density lipoprotein (HDL) cholesterol levels.10 At the cellular level, PPAR agonists act on most cell types involved in atherosclerosis, including endothelial cells, smooth muscle cells (SMCs), macrophages, and lymphocytes, reducing their involvement in the tissue response associated with plaque development. These agonists reduce levels of plasma proteins such as C-reactive protein (CRP), tumor necrosis factor (TNF) {alpha}, and interferon {gamma}11; inhibit IL-2 and TNF{alpha} secretion by monocytes12; and reduce IL-1–induced secretion of IL-6 via nuclear factor (NF) {kappa}B signaling pathways in SMCs.13,14

PPAR agonists have a number of other actions that positively modulate vascular effects. In the endothelium, for example, they inhibit production of the vasoconstrictor endothelin-115,16 and inhibit cytokine-induced expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1.17 In monocyte/macrophages, chemotaxis by monocyte chemotactic peptide-1 and proteolytic enzyme activity by matrix metalloproteinase (MMP)-9 are inhibited,18–20 and the proliferation and migration of SMCs are inhibited.21 Both PPAR{alpha} and PPAR{gamma} stimulate ATP-binding cassette transporter A1 expression, thereby promoting cholesterol efflux from macrophages22 and possibly cholesterol excretion into the gut.

In the clinical setting, several studies with PPAR{alpha} agonists have shown significant reduction in cardiovascular disease risk, especially in subjects with insulin resistance.23–25 In the recently completed FIELD trial, fenofibrate treatment significantly reduced the need for coronary revascularization, but despite this the primary end point for the study was not met.26 PPAR{gamma} agonists have also been shown to reduce the progression of intima-media thickening in patients with coronary artery disease,27 and recent evidence suggests that pioglitazone reduces the incidence of myocardial infarction and stroke in patients with type 2 diabetes and pre-existing cardiovascular disease, although again the primary end point of this study was not met.28

Dual PPAR{alpha}/{gamma} agonists (such as tesaglitazar, which until recently was in phase III clinical development) have been shown to improve both glucose and lipid abnormalities in animal models and human subjects with insulin resistance and/or type 2 diabetes.29–31 Based on their effects in animal models and humans, it has been proposed that dual PPAR{alpha}/{gamma} agonists may have additional benefits beyond their effect on the associated dyslipidemia in reducing components of insulin resistance that contribute to atherosclerosis and cardiovascular disease.31,32 In this study, we examined whether tesaglitazar could reduce atherosclerosis using ApoE*3Leiden transgenic mice made insulin resistant and dyslipidemic with a high-fat diet.

When fed a high-cholesterol diet, ApoE*3Leiden transgenic mice develop a human-like lipoprotein profile, which includes elevated plasma levels of very-low-density lipoprotein, intermediate density lipoprotein, and low-density lipoprotein (LDL), and leads to the development of atherosclerosis. In addition, when fed a high-calorie, high-fat, high-cholesterol diet, these mice develop insulin resistance.33 Depending on their plasma cholesterol levels, ApoE*3Leiden mice develop atherosclerotic lesions that have comparable morphological, histological, and immunohistochemical characteristics to human lesions.34–36 Because plasma cholesterol levels in ApoE*3Leiden transgenic mice can be titrated to any level by adjusting dietary cholesterol intake, we were able to study the effects of tesaglitazar on atherogenesis, independent of its total plasma cholesterol-lowering effect.


*    Methods
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Animals
Female heterozygous ApoE*3-Leiden transgenic mice (3 to 4 months of age), characterized by an enzyme-linked immunosorbent assay for human apoE,35 were used. Animal experiments were approved by the Institutional Animal Care and Use Committee of The Netherlands Organization for Applied Scientific Research (TNO). Animals were provided by TNO-Biomedical Research (Leiden, The Netherlands).

Diets
During a run-in period of 3 weeks, animals received a high-fat/high-cholesterol diet, containing 23% (wt/wt) bovine lard and 1% (wt/wt) cholesterol (HC diet).35 After the run-in period, mice were matched for age and cholesterol level into 3 groups of 17 mice each. One group maintained the HC diet (HC group). The tesaglitazar-treated group received the same HC diet, but the diet was supplemented with tesaglitazar (0.5 µmol/kg diet), equaling 20 µg/kg body weight per day (T group). Tesaglitazar [(S)-2-Ethoxy-3-[4-[2-(4-methylsulphonyloxyphenyl) ethoxy]phenyl propanoic acid], with an EC50 value for activating mouse PPAR{alpha} of 32 µmol/L and for PPAR{gamma} of 0.25 µmol/L,31 was provided by AstraZeneca R&D, Mölndal, Sweden. The tesaglitazar dose used was selected from a pilot experiment, in which we aimed at only 20% cholesterol reduction to ensure atherosclerosis development within a reasonable time period. The low-cholesterol (LC) group received the same diet as the HC group, but containing 0.1% (wt/wt) instead of 1% cholesterol to titrate the plasma cholesterol level to that of the T group, as deduced from previous experiments in our laboratory. The LC group served as the cholesterol-matched control for the T group. The 3 mice groups were treated for 28 weeks. All animals had free access to food and water. Body weight and food intake were monitored, and blood samples were taken after a 4-hour fast by tail bleeding throughout the study.

Analysis of Plasma
Commercially available kits were used to measure total plasma cholesterol (No. 1489437; Roche Diagnostics) and triglyceride levels (No. 337-B; Sigma Diagnostics). Cholesterol exposure was calculated as the area under the curve of cholesterol levels versus time in weeks. Lipoprotein distribution was determined by fast performance liquid chromatographic (fast protein liquid [FPLC]) size fractionation (Pharmacia).35

Glucose and insulin levels were determined after euthanization at week 28. Plasma glucose was measured using commercial reagents (No. 2319 and 2320; Instruchemie) and plasma insulin was measured using a mouse-specific enzyme-linked immunosorbent assay (10 to 1150–01, Alpco). Homeostasis Model Assessment Insulin Resistance (HOMA-IR), a surrogate measure of insulin resistance, was calculated as the product of fasting insulin (µU/mL) and glucose (mmol/L) concentrations divided by 22.5.37 Plasma fibrinogen (homemade mouse kit)38 and serum amyloid A (SAA; Biosource) were measured using specific enzyme-linked immunosorbent assays.

Analysis of Atherosclerosis
After 28 weeks, animals were euthanized and the hearts were harvested, fixed, and embedded in paraffin.35 Serial 5-µm cross-sections of the entire aortic valve area (AVA) were prepared and stained with hematoxylin-phloxin-saffron (HPS) for histological analysis, and with Sirius Red to quantify the collagen area. Atherosclerotic lesions were categorized into types I to V, as described previously35 (see supplemental Figure I, available online at http://atvb.ahajournals.org). Cross-sectional lesion areas were quantified using Leica Qwin morphometric software.39 Four sections of each specimen, containing 3 valve segments each, were analyzed at 40-µm intervals to determine the average lesion number (numbers were counted and totalled per specimen and finally meaned per group [HC/T/LC]), type, and area.40 In addition, descending aortas were isolated and snap-frozen until further analysis. During later analysis, vessels were cleaned of adherent fat, and then stained for lipids using Oil red O for "en face" morphometry of the atherosclerotic lesion area (Leica Qwin morphometric software). All analyses were performed blind, without previous knowledge of feeding regime or treatment.

The number of monocytes adhering to atherosclerotic plaques may give an indication of endothelial activation, and thereby of the inflammatory status of the plaque. Macrophages were detected using AIA31240 anti-serum (1:3000; Accurate Chemical and Scientific). The inflammatory status of plaques was further examined by estimating the local presence of NF{kappa}B (a major regulatory component of inflammatory reactions) in the plaque. NF{kappa}B was detected using mouse anti-human p65-NF{kappa}B (F-6, 1:100; Santa Cruz Biotechnology). Endothelial and macrophage NF{kappa}B-positive cells (cytoplasm and nuclei) were counted per segment. ICAM-1 and MCP-1 were detected using rat anti-mouse CD54 (CBL1316, 1:150; Chemicon International) and goat anti-mouse MCP-1 (M18, 1:300; Santa Cruz Biotechnology), respectively.

Statistical Methods
Nonparametric Mann–Whitney U tests were used to analyze treatment differences, unless stated otherwise. P<0.05 (2-sided) was considered significant. Frequency data for lesion categorization were compared using the Fisher exact test. All data are presented as mean±SD.


*    Results
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Plasma Lipids and Lipoprotein Profiles
Body weight (Figure 1A) and food intake (data not shown) did not differ between the 3 treatment groups during the study periods. As compared with the HC group, plasma cholesterol levels were reduced in the tesaglitazar-treated (T) group (Figure 1B). As required by the experimental design, the total plasma cholesterol levels were similar in the LC and T groups.


Figure 1
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  		Figure 1. Effect of tesaglitazar on plasma lipids in ApoE3*Leiden mice on a high-fat diet. A, Body weight over time. B, Plasma cholesterol over time. C, Lipoprotein profiles. D, Total cholesterol exposure. E, Plasma triglycerides over time. In the LC group, dietary cholesterol was adjusted at week 20 from 0.1% to 0.5% cholesterol (wt/wt) to remain matched with the T group. Circles=HC; triangles=T; squares=LC; *P<0.05

Lipoprotein profiles of the mice showed that tesaglitazar decreased very-low-density lipoprotein cholesterol levels. Additionally, after tesaglitazar treatment, a lipoprotein fraction appeared with a size between LDL and HDL lipoproteins (Figure 1C). Western blot analysis revealed that this lipoprotein fraction was poor in apoAI and apoB but rich in apoE (data not shown).

As derived from the area under the curve of Figure 1B, the HC group had significantly increased exposure to cholesterol compared with the respective T and LC groups (Figure 1D). There was no significant difference in cholesterol exposure between T and LC control groups. Triglyceride levels were significantly lower in T groups compared with HC and LC groups (Figure 1E) (P<0.05).

In the T group, plasma tesaglitazar levels reached 38.6±11.4 nmol/L.

The HOMA-IR index indicated insulin resistance in HC mice at 28 weeks. HOMA-IR was significantly lower in both the T and LC groups compared with the HC group (4.3±3.2 versus 3.4±2.7 versus 11.1±6.8, respectively; P<0.05).

Atherosclerosis
Cross-sections of the AVA showed that tesaglitazar reduced atherosclerosis compared with the respective HC groups and cholesterol-matched LC groups (Figure 2A). Treatment with tesaglitazar significantly reduced total lesion area by 92% compared with the HC group, and by 83% compared with the cholesterol-matched LC control group (P<0.05). Oil-red-O–stained en-face preparations of the aortic arch and thoracic aorta show a similar trend, but because of the large standard deviation did not reach statistical difference (supplemental Figure II).


Figure 2
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  		Figure 2. Effect of tesaglitazar on atherosclerosis in the aorta of ApoE3*Leiden mice on a high-fat diet. A, The cross-sectional lesion area in the aortic valve area. B, Total number of lesions. C, Lesion severity. *P<0.05

In the same cross-sections we counted the number of lesions. The average number of lesions per animal did not differ significantly between the HC and LC control groups (Figure 2B). However, treatment with tesaglitazar significantly reduced the average number of lesions by 73% compared with the HC group and by 67% compared with the cholesterol-matched LC group (P<0.05). When lesions were categorized as either mild or severe, there was a significant shift (P<0.05) from severe to mild lesions in tesaglitazar-treated animals (Figure 2C).

To further characterize the atherosclerotic lesions, we measured macrophage and collagen areas in cross-sections serial to those used for morphometry (Figure 3). The macrophage-positive area was larger in the HC group compared with the tesaglitazar and LC groups (Figure 3A, 3B). Moreover, the macrophage-positive area was significantly smaller in the T group than in the cholesterol-matched LC group. The collagen-positive areas followed a similar trend (Figure 3A, 3C). When expressed as a percentage of the total lesion area, collagen decreased and macrophages increased in accordance with the shift to less severe lesions.


Figure 3
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  		Figure 3. Cross-sectional lesion characteristics. A, Representative microscopic images of macrophage and collagen staining. B, Macrophage area. C, Collagen area. *P<0.05; scale bar= 100 µm.

Inflammatory Markers
SAA levels were significantly reduced (P<0.05) in T and LC groups compared with HC group (–50.5% and –23%, respectively; Figure 4A), tesaglitazar being more efficient than LC in this respect. Fibrinogen levels were unaffected (data not shown). There were fewer adhering monocytes in the tesaglitazar-treated group compared with the HC group (Figure 4B). There were no differences between the tesaglitazar-treated group and the cholesterol-matched LC control group in this respect.


Figure 4
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  		Figure 4. Effect of tesaglitazar on inflammatory parameters in ApoE3*Leiden mice on a high-fat diet. A, Plasma SAA level. B, Monocyte adherence. *P<0.05.

P65-NF{kappa}B staining was found in the cytosol and nuclei of both endothelial cells and macrophages (Figure 5A). SMCs remained unstained. Positively stained endothelial cells were observed on plaques. When observed on normal vessel walls, the positively stained cells were in close proximity to the shoulder regions of plaques. Cytosolic and nuclear P65-NF{kappa}B expression were quantified in endothelial cells and macrophages (Figure 5B, 5C). In the endothelium, cytosolic as well as nuclear NF{kappa}B expression was decreased in both the tesaglitazar-treated and LC-treated group as compared with the HC-treated group. There were no differences between the tesaglitazar-treated group and the LC control group in this respect (Figure 5B). Macrophage NF{kappa}B expression was reduced in the cytosol and nucleus of the tesaglitazar and LC group compared with the HC-treated group. However, tesaglitazar further decreased the number of cytosolic NF{kappa}B expressing cells when compared with the LC-treated group (Figure 5C). HC- and LC-treated mice had NF{kappa}B expression in endothelial cells and macrophages of almost all segments; however, in the tesaglitazar-treated group, only 17/45 segments showed little endothelial NF{kappa}B positive staining and 1/45 segments showed macrophage endothelial NF{kappa}B-positive staining. We calculated the relative nuclear NF{kappa}B expression, using only the mice that showed NF{kappa}B expression in their aortic segments. In the endothelial cells and macrophages of the HC-treated group 10.2% and 12.7% of the NF{kappa}B positive staining was nuclear, ie, active NF{kappa}B, respectively. In the LC-treated group 7.6% in the endothelium and 3.8% in the macrophages was active NF{kappa}B.


Figure 5
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  		Figure 5. Effect of tesaglitazar on inflammatory markers in atherosclerotic plaques of ApoE3*Leiden mice on a high-fat diet. A, Representative microscopic pictures of p65-NF{kappa}B–positive staining of atherosclerotic plaques. B, Scoring of endothelial. C, Macrophage cytosolic (black bars) and nuclear (white bars) NF{kappa}B-positive staining. D, Representative microscopic pictures of ICAM-1. E, MCP-1–positive staining.*Cytosol and {dagger}nucleus P<0.05 (scale bar=100 µm).

ICAM-1 staining was found on endothelial cells. Although staining was present in segments with and without lesions, the endothelial lining of ICAM-1 expression appeared less intense in segments without lesions (ie, the tesaglitazar treated group) (Figure 5D).

MCP-1 staining was found in macrophages/foam cells and endothelial cells. Because the lesions in the tesaglitazar treated group were smaller less MCP-1–positive staining was observed in this group, compared with the HC and LC groups (Figure 5E).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study showed that tesaglitazar has atherosclerosis-reducing capacities in ApoE*3Leiden transgenic mice that cannot be attributed solely to its reduction of plasma total cholesterol. The mechanism by which tesaglitazar reduced atherogenesis in these mice could therefore involve changes in lipoprotein composition, and thereby quality, in combination with direct actions on the pro-inflammatory tissue response of vascular cells.

The hyperlipidemic ApoE*3Leiden mice used here have a lipoprotein profile that is more similar to the human profile than those of either apoE–/– or LDLr–/– mice. In agreement with previous studies with ApoE*3Leiden mice,34,35,41 we were able to titrate plasma cholesterol levels by adjusting dietary cholesterol intake. Previous studies have also shown that these mice respond to hypolipidemic drugs; treatment with statins reduces plasma cholesterol42,43 and treatment with a PPAR{alpha} agonist reduces both plasma cholesterol and triglyceride levels (unpublished data). In addition, we showed in an earlier dose-finding study that ApoE*3Leiden mice respond to the dual PPAR{alpha}/{gamma} agonist tesaglitazar. In the present study, we aimed for mild cholesterol-lowering with tesaglitazar to investigate direct anti-atherosclerotic effects on the vascular wall. At a dose of tesaglitazar 0.5 µmol/kg diet (or 20 µg/kg body weight/d), a moderate decrease in plasma cholesterol {approx}20% was achieved.

The lipoprotein profiles of the mice suggested that treatment with tesaglitazar resulted in the formation of an additional particle, sized between the LDL and HDL fractions. Western blotting characterized the particle as poor in apoAI and apoB but rich in apoE (data not shown). Similar lipoprotein profiles have been observed after treatment of ApoE*3Leiden mice with the PPAR{alpha} agonist fenofibrate (unpublished data). Because cholesteryl ester transfer protein is not expressed in mice, these particles could represent large apoE-rich HDL.44 Furthermore, the appearance of these large apoE-rich particles during tesaglitazar treatment was associated with a decrease in atherosclerosis, suggesting that they may have favorable anti-atherosclerotic properties. However, it remains unclear whether the accumulation of these particles is clinically relevant or a mouse-specific effect.

To examine the effects of tesaglitazar that might contribute to a reduction in atherosclerosis additional to the observed lipoprotein changes, we analyzed the levels of anti-inflammatory markers SAA and fibrinogen in plasma, and examined adhering monocytes and vascular NF{kappa}B expression in atherosclerotic plaques. We found a decrease in plasma SAA levels for tesaglitazar-treated mice, but no change in plasma fibrinogen levels. In tesaglitazar-treated mice, fewer monocytes adhered to the endothelium over plaques, coinciding with a decreased NF{kappa}B expression at the same location. Previous studies have shown evidence for anti-inflammatory activities of PPAR{alpha} and PPAR{gamma} agonists, caused by upregulation of I{kappa}B, leading to decreased NF{kappa}B/C-EBPß complexes and suppression of C-reactive protein synthesis.45 Our model provides further evidence of the anti-inflammatory effects of tesaglitazar, including reduced total lesion area as a result of decreased relative macrophage and collagen areas. Both effects contributed to the observed decrease in plaque severity in drug-treated animals. These anti-inflammatory effects were more pronounced in the tesaglitazar groups than in the LC groups, and were thus not caused by cholesterol-lowering per se. Instead of HF feeding conditions, we performed the same study under low-fat (LF) feeding conditions. The effects of tesaglitazar under LF conditions on cholesterol-lowering, atherosclerosis, and inflammation were largely the same, though less prominent, as under HF conditions.

In cell-based reporter gene assays with only the ligand-binding domain of the transcription factors, tesaglitazar appeared to be >100-fold more efficacious for mouse PPAR{gamma} than for mouse PPAR{alpha}.31 However, we do not know in the intact animal if changes observed in lipid metabolism, insulin sensitivity, and apparent anti-inflammatory markers can be ascribed to primary individual or combined activation of the 2 transcription factors or if they are secondary to improvement on fatty acid metabolism and modification of endogenous ligands.10

In summary, the dual PPAR{alpha}/{gamma} agonist tesaglitazar showed significant anti-atherogenic effects in this mouse model with moderate insulin resistance. These effects could result from tesaglitazar-induced effects in the lipoprotein profiles and from direct anti-inflammatory and anti-atherosclerotic actions in the vascular wall.


*    Acknowledgments
 
We thank the technicians of TNO-Biomedical Research and AstraZeneca-Sweden for their excellent technical assistance.

Source of Funding

This study was supported by the Netherlands Organization of Scientific Research (NWO/ZonMw grant no. 902-26-242), the Netherlands Heart Foundation (grant no. 2000.051), and AstraZeneca, Mölndal, Sweden. J.W.J. is a clinical established investigator of the Netherlands Heart Foundation (2001D032).

Disclosures

None.


*    Footnotes
 
Original received February 14, 2006; final version accepted August 10, 2006.


*    References
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