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European Heart Journal Advance Access published online on December 6, 2007
European Heart Journal, doi:10.1093/eurheartj/ehm540
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

Rosuvastatin increases vascular endothelial PPAR{gamma} expression and corrects blood pressure variability in obese dyslipidaemic mice

Fanny Desjardins1, Belaïd Sekkali1, Wim Verreth2, Michel Pelat1, Dieuwke De Keyzer2, Ann Mertens2, Graham Smith3, Marie-Christine Herregods4, Paul Holvoet2 and Jean-Luc Balligand1,*

1 Unit of Pharmacology and Therapeutics, Université catholique de Louvain, Belgium
2 Atherosclerosis and Metabolism Unit, Department of Cardiovascular Diseases, Katholieke Universiteit, Leuven, Belgium
3 AstraZeneca, Macclesfield, Cheshire, UK
4 Division of Cardiology, Katholieke Universiteit, Leuven, Belgium

Received 3 November 2006; revised 22 October 2007; accepted 29 October 2007.

* Corresponding author: UCL-FATH 5349, Vésale+5, 52 Avenue Mounier, 1200 Brussels, Belgium. Tel: +32 2 764 5268, Fax: +32 2 762 5269. Email: balligand{at}mint.ucl.ac.be


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Aims: Statins improve atherosclerotic diseases through cholesterol-reducing effects. Whether the latter exclusively mediate similar benefits, e.g. on hypertension, in the metabolic syndrome is unclear. We examined the effects of rosuvastatin on the components of this syndrome, as reproduced in mice doubly deficient in LDL receptors and leptin (DKO).

Methods and results: DKO received rosuvastatin (10 mg/kg/day or 20 mg/kg/day) or saline for 12 weeks. Saline-treated DKO mice had elevated blood pressure (BP) and nitric oxide-sensitive BP variability recorded by telemetry. Compared with saline, rosuvastatin (20 mg/kg/day) had no effect on weight gain and a minor effect on plasma cholesterol. Despite incomplete correction of insulin sensitivity, rosuvastatin fully corrected BP and its variability (P = 0.01), in conjunction with upregulation of PPAR{gamma} (but not PPAR{alpha}) in the aortic arch. Rosuvastatin similarly increased PPAR{gamma} (P = 0.002) and SOD1 (P = 0.01) expression in isolated endothelial cells. Both GW9662, a PPAR{gamma}-specific antagonist, and siRNA raised against PPAR{gamma} abrogated rosuvastatin's effect, which was reproduced in PPAR{gamma}- (but not PPAR{alpha}-) dependent transactivation assays.

Conclusion: Beyond partial improvement in insulin sensitivity, rosuvastatin normalized BP homeostasis in obese dyslipidaemic mice independently of changes in body weight or plasma cholesterol. Upregulation of PPAR{gamma} and SOD1 in the endothelium may be involved as a unique vasculoprotective effect of statin treatment.

Key Words: Statin • Blood pressure • Nitric oxide • Superoxide dismutase • PPAR{gamma}


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Obesity, hypertension, dyslipidaemia, and insulin resistance are clustered in the metabolic syndrome, a predisposing condition for atherosclerotic cardiovascular disease. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) potently reduce cholesterol levels, which largely accounts for the reduction of morbidity and mortality in hypercholesterolaemic patients.1 Statins also reduce cardiovascular disease risk in patients with the metabolic syndrome,2 possibly through additional effects on inflammation.3 Indeed, statins may exert protective effects beyond cholesterol lowering, but their relevance to the clinical benefit of statin treatment is still being debated.

Among the components of the metabolic syndrome, hypertension is commonly preceded by the development of insulin resistance, which has been shown recently to be linked to the production of cellular oxidant radicals.4 Glitazones, or PPAR{gamma}-activating ligands, are widely used as insulin sensitizers and also reduce high blood pressure (BP)5,6 and vascular oxidative stress.7 Likewise, several statins have been shown to improve insulin sensitivity in animal models and patients,3,8 and similarly exhibit anti-inflammatory, as well as BP-reducing effects, which are partly independent of their cholesterol-lowering effects.9,10 Although inhibition of pro-oxidant enzymatic systems, such as NADPH oxidase, has been well documented,11 additional mechanisms may be at play. In particular, superoxide dismutases (SODs) represent a major antioxidant defence system in the vasculature and the Cu/Zn cytosolic SOD (encoded by SOD1), which is largely represented in the endothelium12 and has been shown to be regulated by PPARs in vitro.13 Whether statins may improve BP homeostasis by regulating the expression of SOD1 in endothelial cells, particularly through PPAR{gamma} activation, is however unknown.

To resolve these questions, we studied the effect of rosuvastatin in mice with combined leptin and low-density lipoprotein receptor (LDLR) deficiency that develop all the features of the human metabolic syndrome.14,15 Their expected resistance to the cholesterol-lowering effect of statins allowed us to examine ancillary effects of rosuvastatin on vascular function independent of decreases in plasma cholesterol. In particular, the variability of BP was analysed by telemetry in vivo as a surrogate index of endothelial function and vascular stiffness, both key components of cardiovascular prognosis16 also known to be profoundly influenced by vascular oxidant status.17


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Experimental protocol
DKO mice with both leptin (Ob/Ob) and LDLR deficiency (LDLR–/–) were obtained by crossing LDLR–/– and Ob/+ mice as previously described.15 Experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee. Twelve-week-old mice were injected subcutaneously with rosuvastatin (10 mg/kg/day, n = 6 and 20 mg/kg/day, n = 7) or vehicle (n = 9) for 12 weeks and compared with age-matched (24 weeks) control mice (wild type, WT; C57BL6). Mice were identified by a number and independently assigned to the different experimental groups to avoid selection bias. The results were analysed blindly.

Circadian variation and frequency analysis of blood pressure and heart rate by implanted telemetry
BP signals [and heart rate (HR), derived from pressure waves] from the aortic arch were measured in conscious, unrestrained animals with surgically implanted, miniaturized telemetry devices (Datascience Corp., USA) as described.18

Cell culture and transient transfection
Bovine aortic endothelial cells and HEK293 were cultured to confluence in EGM-MV or DMEM containing 10% serum, then serum-starved for 24 h and exposed to the different treatments. Transient transfection of HEK293 was carried out in 24-well plates at 40–50% confluency (see Supplementary material online for details).

mRNA and protein analysis
mRNA expression in the aortic arch and in aortic endothelial cells was measured by reverse transcription (RT)–real-time quantitative polymerase chain reaction (PCR) as described.15 SOD1 protein expression was measured by western blotting (see Supplementary material online).

Biochemical analysis
Blood was collected from conscious mice by tail bleeding into EDTA tubes after an overnight fast, and biochemical parameters measured as described before15 (see Supplementary material online). The data reported in Table 1 were obtained from those mice that underwent the full telemetry protocol.


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  		Table 1 Blood and metabolic parameters

 
Statistical analysis
For in vivo experiments on BP and BP variability, two different analyses were made. For the assessment of the effect of placebo or two doses of rosuvastatin, the data were analysed by a trend test. To assess whether results after the administration of rosuvastatin were similar to those observed in age-matched C57BL6 mice, we performed one-way ANOVA followed by the Dunnett test. To evaluate the influence of treatment group on the circadian variation, a two-way ANOVA was performed, including an interaction term for treatment by time. All statistical tests were two-sided. For the analysis of the mean 24 h values of haemodynamic parameters, t-test was performed. The sample size (i.e. number of animals per group) was decided from our previous experience with highly accurate and reproducible measurements with telemetry.15 For in vitro experiments, we performed one-way ANOVA with post hoc analysis using the Bonferroni procedure for selected comparisons as indicated.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 
Weight and metabolic parameters
Compared with placebo, rosuvastatin had no effect on weight gain. The metabolic parameters in the different groups are shown in Table 1. All pre-treatment values were identical between placebo- and rosuvastatin-treated animals (data not shown). Rosuvastatin had minimal effects on plasma total cholesterol levels. Conversely, it produced a significant reduction in triglycerides at both doses of the drug. The same doses also decreased glucose and insulin with an improvement in glucose tolerance (Table 1).

Rosuvastatin normalized systolic blood pressure and decreased heart rate in LDLR–/–/ObOb mice
Figure 1 represents the BP and HR values obtained by telemetry over 24 h in the different groups. Night and day values are also displayed in Table 2. Compared with age-matched C57BL6 mice (WT), placebo DKO mice had a significant increase in their mean 24 h systolic BP (SBP, 126.7 ± 2.9 vs. 114.7 ± 2.9 mmHg; P = 0.0007), diastolic BP (DBP, 94.7 ± 3.5 vs. 85.8 ± 3.7 mmHg; P = 0.0008), and HR (547.8 ± 20.4 vs. 442.6 ± 31.5 b.p.m.; P = 0.0001), as well as abolition of their circadian variation of SBP, as measured by two-way ANOVA between WT and placebo (Figure 1A; P = 0.002). Rosuvastatin (10 and 20 mg/kg/day) decreased mean values of SBP in DKO (111.1 ± 5.6 and 115.3 ± 5.1 for R10 and R20, respectively; P = 0.002) and restored the physiological circadian variation of SBP (P = 0.02 and P = 0.01 for R10 and R20, respectively, vs. placebo).


Figure 1
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  		Figure 1 Rosuvastatin corrects circadian variation of blood pressure and heart rate in low-density lipoprotein receptor/ObOb-deficient mice. Circadian variation of blood pressure and heart rate in wild type mice (n = 10) and DKO mice treated with placebo for 12 weeks (Placebo, n = 9) and DKO mice treated for 12 weeks with 10 mg/kg of rosuvastatin (R10, n = 6). Time course of systolic blood pressure (A), diastolic blood pressure (B), and heart rate (C) over 24 h. Shaded areas on the x-axis represent dark cycles (activity period in mice). Mean values (±SEM) of systolic blood pressure, diastolic blood pressure, and heart rate were calculated for each 60 min sequence of recording during the 24 h period

 

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  		Table 2 Mean, night and day values of systolic blood pressure, diastolic blood pressure, and heart rate in wild type (C57BL/6), DKO mice treated with placebo and rosuvastatin 10 mg/kg/day and 20 mg/kg/day

 
Rosuvastatin restored the nitric oxide-dependent control of blood pressure variability and its sensitivity to nitric oxide synthase inhibition in LDLR–/–/ObOb mice
Spectral analysis of the 24 h SBP recordings was performed, and the variability of SBP (SBPV) in the very low frequency (VLF) band (0.05–0.4 Hz; reflecting neurohumoral control, including nitric oxide, NO) was measured (Figure 2A). Compared with WT mice, the SBPV of placebo DKO mice was higher (62.7 ± 1.3 vs. 54.4 ± 2.1; P = 0.01), suggesting an altered neurohumoral control of BP. Treatment with both 10 and 20 mg/kg rosuvastatin resulted in a similarly decreased index of SBPV in the VLF domain in DKO mice compared with the placebo group (44.7 ± 1.9 and 44.6 ± 1.0, respectively, P = 0.01). The VLF values of both rosuvastatin-treated groups were even lower than in the WT (P = 0.01). To verify the involvement of the NO component in the altered control of variability in the VLF, the different groups of mice were subjected to a pharmacological test with an NO synthase (NOS) inhibitor, and the sensitivity of their variability index in the VLF compared (Figure 2B). As expected, acute inhibition of NOS upon injection of the NOS inhibitor L-NAME increased the VLF index in WT mice, as previously shown by us.18 However, this increase was substantially reduced in the placebo DKO mice, suggesting a reduced NO-dependent buffering of their SBPV. Importantly, the L-NAME-sensitivity of the VLF index was restored after treatment with rosuvastatin (at both doses) (Figure 2B). These results suggest that the decreased SBP buffering capacity in DKO mice is indeed NO-dependent and that the beneficial effect of rosuvastatin involves restored production (and/or bioavailability) of NO.


Figure 2
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  		Figure 2 Rosuvastatin corrects nitric oxide-dependent systolic blood pressure variability in low-density lipoprotein receptor/ObOb-deficient mice. Spectral analysis of systolic blood pressure variability in DKO mice treated with placebo (Placebo, n = 9) and DKO mice treated for 12 weeks with 10 mg/kg of rosuvastatin (R10, n = 6) or 20 mg/kg (R20, n = 7) compared with control C57BL6 (WT, n = 10). After normalization to whole power spectra, area under the curve for the variability of systolic blood pressure was calculated for each group. Results are presented for specific frequency bands, i.e. very low frequency of systolic blood pressure variability (0.05–0.4 Hz, reflecting neurohumoral control) (A) and its increase after acute nitric oxide synthase inhibition using intraperitoneal injection of L-NAME (30 mg/kg) (B)

 
Rosuvastatin restored the sympathetic and parasympathetic control of blood pressure and heart rate variability in DKO (LDLR–/–/ObOb) mice
Notably, rosuvastatin also partly normalized the higher SBPV in the low frequency domain (LF, 0.4–1.5 Hz; reflective of the sympathetic tone; P = 0.03, Figure 3A), which was compatible with the decrease in HR as illustrated in Figure 1C; moreover, the adrenergic tone was assessed from the HR response after an acute challenge with propranolol. There was a more pronounced decrease in HR in DKO placebo compared with WT mice (–148.9 ± 18.3 vs. –63.5 ± 15.1 b.p.m.; P = 0.008), which was compatible with an increased basal sympathetic tone in DKO mice. This response was reduced after rosuvastatin treatment (R20: –97.5 ± 9.9 b.p.m.; P = 0.03). Rosuvastatin also partly reversed the decreased variability of HR in the high frequency (HF, 1.5–5.0 Hz; reflective of parasympathetic tone; P = 0.04; Figure 3B), which suggests a restoration of vagal tone, as assessed from the HR response to an acute challenge with atropine. Atropine induced less elevation in HR in DKO placebo compared with WT mice (61.3 ± 23.9 vs. 141.8 ± 12.0 b.p.m.; P = 0.04), confirming the diminished basal control of HR in DKO mice. This reduced control of HR by the parasympathetic nervous system was partly restored after rosuvastatin treatment (R20: 126.2 ± 9.1 b.p.m.; P = 0.04).


Figure 3
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  		Figure 3 Rosuvastatin corrects autonomic control of blood pressure and heart rate in low-density lipoprotein receptor/ObOb-deficient mice. Spectral analysis of systolic blood pressure variability and heart rate variability in DKO mice treated with placebo (Placebo, n = 9) and DKO mice treated for 12 weeks with 10 mg/kg of rosuvastatin (R10, n = 6) or 20 mg/kg (R20, n = 7) compared with control C57BL6 (WT, n = 10). After normalization to whole power spectra, area under the curve for the variability of systolic blood pressure and heart rate was calculated for each group. Results are presented for specific frequency bands, i.e. LF of systolic blood pressure variability (0.4–1.5 Hz, reflective of the adrenergic tone) (A) and high frequency of variability (1.5–5 Hz, reflective of parasympathetic tone) (B)

 
Rosuvastatin increased PPAR{gamma} and SOD1 expression in aortic tissue and endothelial cells
Because of the involvement of the transcription factors PPARs in the control of insulin sensitivity and inflammation, which may determine the development of endothelial dysfunction, the expression of PPARs was examined in extracts of the aortic arch by RT–PCR. Our previous study had shown a decrease of aortic PPAR{gamma} and {alpha} expression in DKO vs. WT mice.15 Here, rosuvastatin restored PPAR{gamma} expression compared with placebo DKO (R10 and R20: 100%; P = 0.007) (Figure 4A), but had no significant effect on PPAR{alpha} expression (R10: 0.62 ± 0.29; R20: 0.49 ± 0.20 vs. placebo: 0.36 ± 0.15; P = 0.09 and 0.23, respectively). Rosuvastatin also increased SOD1 expression in aortic tissue.19


Figure 4
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  		Figure 4 Rosuvastatin increases PPAR{gamma} and SOD1 expressions in aortic tissue and aortic endothelial cells. The expression of PPAR{gamma} was measured in aortic tissue extracts and compared with placebo (n = 9) and rosuvastatin-treated animals (R10, n = 6; R20, n = 7). Results are normalized to the levels in wild type (C57Bl6) animals (A). Bovine aortic endothelial cells were cultured and incubated for a period of 24 h with vehicle (n = 12), 10–5 mol/L of rosuvastatin (n = 11) (B), 5 x 10–6 mol/L of GW9662 (PPAR{gamma} antagonist, n = 6), and 10–5 mol/L of rosuvastatin + 5 x 10–6 mol/L of GW9662 (n = 6) (C). mRNA levels, as measured by real-time polymerase chain reaction (see Methods for technical details) for PPAR{gamma} (B) and SOD1 (C). Results are expressed as a percentage of the control (vehicle) level. Dose-dependent effect of rosuvastatin on SOD1 protein level in bovine aortic endothelial cells (n = 6), compared with {alpha}-actinin as loading control (D)

 
To ascertain a specific effect on the endothelium, we next examined the effect of rosuvastatin on PPAR{gamma} expression in cultured endothelial cells. After 24 h of incubation with rosuvastatin at 10–5 mol/L, expression of PPAR{gamma} mRNA was significantly increased (70% vs. control; P = 0.002) (Figure 4B).

We then measured the expression of Cu/Zn SOD (SOD1) as a target gene for PPAR-mediated transcriptional control that may account for the restored NO-dependent endothelial function, as observed in vivo. Under the same conditions as earlier, rosuvastatin produced a significant increase in SOD1 mRNA expression (43%; P = 0.001) compared with control (Figure 4C). This was confirmed with dose-dependent increases in SOD1 protein levels (Figure 4D).

To verify that the increase in SOD1 expression was a result of PPAR{gamma} transactivation, we also treated cells with GW9662, a PPAR{gamma} antagonist.20 As shown in Figure 4C, GW9662 alone had no significant effect on basal levels of SOD1. However, GW9662 abrogated the upregulation of SOD1 in response to rosuvastatin, supporting the causality of PPAR{gamma} activation on this effect.

We next assessed the effects of the PPAR{gamma} agonist pioglitazone and the PPAR{alpha} agonist WY14643 on PPAR{gamma} and SOD1 expression in endothelial cells. Pioglitazone induced a significant increase in PPAR{gamma} expression (73%, P = 0.04, again inhibited by GW9662, P = 0.001), whereas WY14643 had no effect (P = 0.25). However, as shown in Figure 5A, both pioglitazone and WY14643 induced an increase in SOD1 expression (although more modest with the PPAR{alpha} agonist). GW9662, again, selectively inhibited the effect of pioglitazone only.


Figure 5
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  		Figure 5 Pioglitazone increases SOD1 expression and PPAR{gamma}-targeted siRNA transfection abolishes the upregulation of SOD1 in response to rosuvastatin in aortic endothelial cells. Graphs illustrate mRNA levels, as measured by real-time polymerase chain reaction (see Methods for technical details) for SOD1. (A) Bovine aortic endothelial cells were cultured and incubated for a period of 24 h with vehicle (n = 14), 10–5 mol/L of pioglitazone (PPAR{gamma} agonist, n = 9) alone or with 5 x 10–6 mol/L of GW9662 (PPAR{gamma} antagonist, n = 6), 10–5 mol/L of WY14643 (PPAR{alpha} agonist, n = 6) alone or with 5 x 10–6 mol/L of GW9662 (n = 4). (B) Bovine aortic endothelial cells were cultured and transfected with two different siRNAs targeting PPAR{gamma}. Cells were then incubated for a period of 24 h with vehicle (n = 12 for control, n = 7 for siRNA-1, n = 7 for siRNA-2) or 10–5 mol/L of rosuvastatin (n = 9 for control, n = 9 for siRNA-1, n = 9 for siRNA-2). Results are expressed as a percentage of the control (vehicle) level

 
To gain further proof of the involvement of PPAR{gamma} in rosuvastatin-induced SOD1 upregulation, we used two different siRNAs targeting PPAR{gamma}. Both siRNAs decreased PPAR{gamma} expression (by 66 and 49%, respectively; P = 0.001) and abrogated the increase in PPAR{gamma} (P = 0.09 and 0.21, respectively) as well as SOD1 expression by rosuvastatin (Figure 5B).

Rosuvastatin increased PPAR{gamma}-dependent transactivation of PPAR-responsive genes
Finally, we analysed the effect of rosuvastatin in HEK293 cells transiently transfected with a luciferase reporter construct containing three copies of the PPRE site of the human apolipoprotein A-II promoter flanking the thymidine kinase promoter (PPRE3-TK-Luc). Co-transfection of plasmids encoding PPAR{gamma} or PPAR{alpha} (at low or high amounts) induced promoter activity, which was robustly enhanced by pioglitazone (PPAR{gamma} synthetic agonist) and WY14643 (PPAR{alpha} synthetic agonist), respectively (Supplementary material online, Figure S1). Cell treatment with rosuvastatin enhanced PPAR{gamma}-dependent luciferase activity (P = 0.04) and had a marginal effect on PPAR{alpha} transactivation (P = 0.13). PPAR{gamma} transactivation was again blocked by GW9662 (Figure 6).


Figure 6
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  		Figure 6 Rosuvastatin increases PPAR{gamma}-dependent transactivation of a luciferase reporter construct in HEK293 cells. Cells were co-transfected with plasmids encoding PPAR{gamma} or PPAR{alpha} (55 or 110 ng) and a construct containing three copies of the PPRE site of the human apolipoprotein A-II promoter flanking the thymidine kinase promoter (PPRE3-TK-Luc); a β-galactosidase plasmid was co-transfected in all experiments to correct for transfection efficiency. Cell treatment with rosuvastatin enhanced PPAR{gamma}- (but not PPAR{alpha}-) dependent luciferase activity, an effect, again, blocked by GW9662. Results are expressed as luciferase/β-galactosidase signal ratio (n = 5 independent experiments, all in triplicate)

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary material
 Funding
 Acknowledgements
 References
 
The main findings of our study are that in a mouse model of the human metabolic syndrome, rosuvastatin (i) reduced plasma triglycerides and improved insulin sensitivity, with only a marginal effect on total cholesterol; (ii) corrected high BP and NO-dependent SBPV; (iii) increased PPAR{gamma} expression in the aortic arch in vivo and in cultured endothelial cells in vitro, as well as endothelial SOD1, an antioxidant, PPAR-responsive gene; (iv) enhanced PPAR{gamma}- (but not PPAR{alpha}-) transactivation of a PPAR-responsive reporter gene.

Our analysis showed that the development of high BP was paralleled with increases in triglycerides, glucose, and insulin in DKO mice, consistent with the proposed pathophysiological importance of insulin resistance for the development of hypertension in the metabolic syndrome.6,21 The relationship between insulin resistance and abnormal vascular reactivity has been demonstrated in a variety of clinical conditions, and a reduction in systemic insulin resistance is accompanied by improved endothelial function and vice versa.4,22 Accordingly, the dose-dependent effect of rosuvastatin on systemic insulin resistance may be part of the explanation for the beneficial effect on BP homeostasis in our model.

However, an important observation in this study is that, even at the lower dose, rosuvastatin completely corrected SBP and SBPV despite incomplete correction of insulin resistance. This suggests that additional mechanisms must be at play, perhaps through direct effects on molecular targets in the central nervous system or the vascular wall.9 Indeed, our observation of rosuvastatin's effects on LF and HF variability (Figure 3), reflecting a restoration of the sympathovagal balance, suggests central effects on specific brain nuclei, possibly through inhibition of Rho/Rho-kinase.23 In addition, our frequency analysis of BP tracings indicates an effect on SBPV in the VLF domain, an index of humoral control of vessel tone, including by the vascular relaxant NO.24 We and others have previously validated this parameter as reflecting the ‘buffering’ capacity of NO on SBP in the mouse,18,24 i.e. decreased production/activity of NO results in increased SBPV, as observed in the untreated DKO mice. Regardless of the dose used, rosuvastatin decreased SBPV. That this effect involved a restoration of NO is confirmed by the comparative sensitivity to acute inhibition of NOS with L-NAME (Figure 2B).18 Rosuvastatin's effect on variability is unlikely to be only the consequence of SBP normalization, because variability was lowered to levels below those observed in wild-type animals (Figure 2A) at similar-day SBP levels (Figure 1A and Table 2). Therefore, a direct effect on vascular NO is more likely to be the cause, rather than the consequence of BP correction.

Several mechanisms may account for such direct vascular effects. In addition to upregulation of endothelial NO synthase (eNOS) expression or activity,25,26 rosuvastatin may restore vascular NO signalling by increasing NO bioavailability (for a review, see Pelat and Balligand9). A prominent factor influencing the latter is the prevailing oxidative stress in the vascular wall. PPAR{gamma} exerts well-established anti-inflammatory effects through both transcriptional regulation and trans-repressional effects on key pro-inflammatory and pro-oxidant signalling pathways, such as NF-kappaB.27,28 Accordingly, rosuvastatin upregulated PPAR{gamma} mRNA expression in the aortic arch of our telemetered mice, as well as in isolated endothelial cells (Figure 4A and B), which are known to express this PPAR isoform.13 Statins can activate the transcription of PPAR{gamma} through a SREBP response element in the PPAR{gamma} promoter.29 However, this does not necessarily translate into increased PPAR{gamma} protein abundance (not directly measured here), which was previously shown to be reduced by ligand binding as a result of receptor degradation.30 This does not exclude activation of PPAR{gamma} transcriptional activity by the statin, as illustrated in our transactivation assay. Of note, the expression and activity of endothelial PPAR{gamma} have been causally linked with BP regulation independently of changes in systemic insulin sensitivity.31 Our observations in vivo now provide an additional mechanism for the correction of endothelial dysfunction by rosuvastatin beyond its effects on systemic insulin resistance. The fact that, compared with glitazones,32 rosuvastatin has a more prominent BP-lowering effect suggests that statins probably activate additional mechanisms beyond PPAR{gamma} activation.

Among PPAR-regulated genes with known antioxidant effects, we found the copper–zinc-containing SOD (Cu/Zn SOD or SOD1) to be upregulated in isolated endothelial cells (Figure 4). This cytosolic enzyme represents the predominant SOD in the vasculature and is abundantly expressed in the endothelium.12 The SOD1 gene promoter contains a PPAR-response element that mediates its induction by PPARs and may contribute to the antioxidant effects of PPAR{gamma} agonists in the endothelium.13 Accordingly, both GW9662, a PPAR{gamma}-specific antagonist, and cell transfection with siRNA raised against PPAR{gamma} abrogated the upregulation of SOD1 in response to rosuvastatin, demonstrating the causal involvement of PPAR{gamma} in the upregulation of SOD1 (Figures 4C and 5B). Furthermore, in transactivation assays using co-transfection of plasmids encoding PPAR{gamma} or PPAR{alpha} with a luciferase reporter construct under the control of PPAR-responsive elements, cell treatment with rosuvastatin enhanced PPAR{gamma}- (but not PPAR{alpha}-) dependent luciferase activity, an effect, again, selectively blocked by GW9662. The ensuing protection of NO from scavenging by superoxide anions likely participates in the restoration of NO-dependent endothelial function and BP regulation. SOD1 also protects against oxidant-mediated vascular smooth muscle hyperplasia and hypertrophy, two key pathogenic factors for vascular stiffness that are directly correlated with increased SBPV17 and were recently shown to be attenuated by pitavastatin in hypercholesterolaemic rabbits.33 Finally, insulin resistance has recently been shown to be mechanistically linked with the generation of cellular ROS,4 so rosuvastatin's effect on PPAR{gamma} and SOD1 may provide a unifying link for the restoration of both NO-dependent endothelial BP control and endothelial insulin sensitivity.

Previous studies indicate that statins activate PPAR{alpha} through a molecular mechanism implicating the geranylgeranyl-pyrophosphate pathway and prenylation of Rho family proteins.34 Moreover, it has been shown that the anti-inflammatory effect of simvastatin occurs via PPAR{alpha} by a mechanism involving inhibition of PKC{alpha} inactivation of PPAR{alpha} transrepression activity in murine macrophages and neutrophils.35 As SOD1 was also upregulated by a PPAR{alpha} agonist in our endothelial cells, rosuvastatin may have exerted some of its effects through PPAR{alpha} as well; however, rosuvastatin had no effect on PPAR{alpha} expression in aortic tissue and marginally affected PPAR{alpha}-mediated transactivation, suggesting a more prominent effect through PPAR{gamma} with this statin.

Clinical significance
A recent analysis comparing the effect of high vs. low dose of statin treatment in patients with the metabolic syndrome and stable coronary disease showed a benefit of the higher dose irrespective of the presence of glycaemic abnormalities,2 suggesting this effect to be at least in part independent of improvements in glucose homeostasis, as in our mouse model. Contrary to this clinical study, where most of the benefit was attributed to incremental cholesterol lowering, rosuvastatin corrected haemodynamics in the absence of major changes in plasma cholesterol in our obese, LDLR-deficient mice, lending support for direct vascular effects of the drug, as suggested for statins in acute coronary syndromes.36 Our demonstration of endothelial PPAR{gamma} and SOD1 upregulation with rosuvastatin, resulting in decreased oxidative stress and improved endothelial dysfunction, adds to the expected benefits in high-risk patients, particularly through the decrease in vascular stiffness and SBPV, both independent predictors of cardiovascular morbidity and mortality.16,17

In conclusion, despite incomplete correction of insulin sensitivity, rosuvastatin normalized BP regulation in DKO mice in the absence of major change in plasma cholesterol. The increase in PPAR{gamma} expression and activity as well as of SOD1 (a PPAR-responsive gene) observed in vivo in aorta and in vitro in endothelial cells may represent a unique mechanism of vascular protection and BP correction by statin treatment.


   Supplementary material
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
Funding
 Acknowledgements
 References
 
Supplementary material is available at European Heart Journal online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
Acknowledgements
 References
 
This work was supported by the Interuniversity Attraction Poles Program (P6/30) from the Belgian Science Policy, the FP6-IP018833 ‘EUGeneHeart’, The Fonds National de la Recherche Scientifique, the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (G027604), the Fondation Leducq, and a grant from AstraZeneca. F.D. is Aspirant of the Fonds National de la Recherche Scientifique, Belgium.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
References
 
We thank Dr Marc van Bilsen (University of Maastricht, NL) for supplying plasmid constructs used in the transactivation assays, and Mrs Anne Danniau for help with the statistical analysis. The authors thank Delphine DeMulder, Hilde Bernar, and Michèle Landeloos for technical assistance.

Conflict of interest: J.-L.B. reports having received research grant support from AstraZeneca, and having served on paid advisory boards from AstraZeneca. G.S. is full time employee of AstraZeneca, UK.


   Footnotes
 
This paper was guest edited by Dr Nikolaus Marx, Department of Internal Medicine II-Cardiology, University of Ulm, Germany


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 Funding
 Acknowledgements
 References
 

  1. Baigent C, Keech A, Kearney PM, Blackwell L, Buck G, Pollicino C, Kirby A, Sourjina T, Peto R, Collins R, Simes R. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet (2005) 366:1267–1278.[CrossRef][Web of Science][Medline]
  2. Deedwania P, Barter P, Carmena R, Fruchart JC, Grundy SM, Haffner S, Kastelein JJ, LaRosa JC, Schachner H, Shepherd J, Waters DD. Reduction of low-density lipoprotein cholesterol in patients with coronary heart disease and metabolic syndrome: analysis of the Treating to New Targets study. Lancet (2006) 368:919–928.[CrossRef][Web of Science][Medline]
  3. Lundbye JB, Thompson PD. Statin use in the metabolic syndrome. Curr Atheroscler Rep (2005) 7:17–21.[CrossRef][Medline]
  4. Houstis N, Rosen ED, Lander ES. Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature (2006) 440:944–948.[CrossRef][Medline]
  5. Sarafidis PA, Lasaridis AN. Actions of peroxisome proliferator-activated receptors-gamma agonists explaining a possible blood pressure-lowering effect. Am J Hypertens (2006) 19:646–653.[CrossRef][Web of Science][Medline]
  6. Walcher D, Marx N. Insulin resistance and cardiovascular disease: the role of PPARgamma activators beyond their anti-diabetic action. Diab Vasc Dis Res (2004) 1:76–81.[Abstract/Free Full Text]
  7. Mehta JL, Hu B, Chen J, Li D. Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation. Arterioscler Thromb Vasc Biol (2003) 23:2203–2208.[Abstract/Free Full Text]
  8. Huptas S, Geiss HC, Otto C, Parhofer KG. Effect of atorvastatin (10 mg/day) on glucose metabolism in patients with the metabolic syndrome. Am J Cardiol (2006) 98:66–69.[CrossRef][Web of Science][Medline]
  9. Pelat M, Balligand JL. Statins and hypertension. Semin Vasc Med (2004) 4:367–375.[CrossRef][Medline]
  10. 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]
  11. Wassmann S, Laufs U, Muller K, Konkol C, Ahlbory K, Baumer AT, Linz W, Bohm M, Nickenig G. Cellular antioxidant effects of atorvastatin in vitro and in vivo. Arterioscler Thromb Vasc Biol (2002) 22:300–305.[Abstract/Free Full Text]
  12. Faraci FM, Didion SP. Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol (2004) 24:1367–1373.[Abstract/Free Full Text]
  13. Inoue I, Goto S, Matsunaga T, Nakajima T, Awata T, Hokari S, Komoda T, Katayama S. The ligands/activators for peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma increase Cu2+, Zn2+-superoxide dismutase and decrease p22phox message expressions in primary endothelial cells. Metabolism (2001) 50:3–11.[CrossRef][Web of Science][Medline]
  14. Hasty AH, Shimano H, Osuga J, Namatame I, Takahashi A, Yahagi N, Perrey S, Iizuka Y, Tamura Y, Amemiya-Kudo M, Yoshikawa T, Okazaki H, Ohashi K, Harada K, Matsuzaka T, Sone H, Gotoda T, Nagai R, Ishibashi S, Yamada N. Severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor. J Biol Chem (2001) 276:37402–37408.[Abstract/Free Full Text]
  15. Verreth W, De Keyzer D, Pelat M, Verhamme P, Ganame J, Bielicki JK, Mertens A, Quarck R, Benhabiles N, Marguerie G, Mackness B, Mackness M, Ninio E, Herregods MC, Balligand JL, Holvoet P. Weight-loss-associated induction of peroxisome proliferator-activated receptor-alpha and peroxisome proliferator-activated receptor-gamma correlate with reduced atherosclerosis and improved cardiovascular function in obese insulin-resistant mice. Circulation (2004) 110:3259–3269.[Abstract/Free Full Text]
  16. Sega R, Corrao G, Bombelli M, Beltrame L, Facchetti R, Grassi G, Ferrario M, Mancia G. Blood pressure variability and organ damage in a general population: results from the PAMELA study (Pressioni Arteriose Monitorate E Loro Associazioni). Hypertension (2002) 39:710–714.[Abstract/Free Full Text]
  17. Zieman SJ, Melenovsky V, Kass DA. Mechanisms, pathophysiology, and therapy of arterial stiffness. Arterioscler Thromb Vasc Biol (2005) 25:932–943.[Abstract/Free Full Text]
  18. Pelat M, Dessy C, Massion P, Desager JP, Feron O, Balligand JL. Rosuvastatin decreases caveolin-1 and improves nitric oxide-dependent heart rate and blood pressure variability in apolipoprotein E–/– mice in vivo. Circulation (2003) 107:2480–2486.[Abstract/Free Full Text]
  19. Verreth W, De Keyzer D, Davey PC, Geeraert B, Mertens A, Herregods MC, Smith G, Desjardins F, Balligand JL, Holvoet P. Rosuvastatin restores superoxide dismutase expression and inhibits accumulation of oxidized LDL in the aortic arch of obese dyslipidemic mice. Br J Pharmacol (2007) 151:347–355.[CrossRef][Web of Science][Medline]
  20. Polikandriotis JA, Mazzella LJ, Rupnow HL, Hart CM. Peroxisome proliferator-activated receptor gamma ligands stimulate endothelial nitric oxide production through distinct peroxisome proliferator-activated receptor gamma-dependent mechanisms. Arterioscler Thromb Vasc Biol (2005) 25:1810–1816.[Abstract/Free Full Text]
  21. Caglayan E, Blaschke F, Takata Y, Hsueh WA. Metabolic syndrome-interdependence of the cardiovascular and metabolic pathways. Curr Opin Pharmacol (2005) 5:135–142.[CrossRef][Web of Science][Medline]
  22. Natali A, Baldeweg S, Toschi E, Capaldo B, Barbaro D, Gastaldelli A, Yudkin JS, Ferrannini E. Vascular effects of improving metabolic control with metformin or rosiglitazone in type 2 diabetes. Diabetes Care (2004) 27:1349–1357.[Abstract/Free Full Text]
  23. Ito K, Hirooka Y, Sakai K, Kishi T, Kaibuchi K, Shimokawa H, Takeshita A. Rho/Rho-kinase pathway in brain stem contributes to blood pressure regulation via sympathetic nervous system: possible involvement in neural mechanisms of hypertension. Circ Res (2003) 92:1337–1343.[Abstract/Free Full Text]
  24. Stauss HM, Godecke A, Mrowka R, Schrader J, Persson PB. Enhanced blood pressure variability in eNOS knockout mice. Hypertension (1999) 33:1359–1363.[Abstract/Free Full Text]
  25. Laufs U, La FV, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation (1998) 97:1129–1135.[Abstract/Free Full Text]
  26. Feron O, Dessy C, Desager JP, Balligand JL. Hydroxy-methylglutaryl-coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance. Circulation (2001) 103:113–118.[Abstract/Free Full Text]
  27. Blaschke F, Takata Y, Caglayan E, Law RE, Hsueh WA. Obesity, peroxisome proliferator-activated receptor, and atherosclerosis in type 2 diabetes. Arterioscler Thromb Vasc Biol (2006) 26:28–40.[Abstract/Free Full Text]
  28. Pascual G, Fong AL, Ogawa S, Gamliel A, Li AC, Perissi V, Rose DW, Willson TM, Rosenfeld MG, Glass CK. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature (2005) 437:759–763.[CrossRef][Medline]
  29. Fajas L, Schoonjans K, Gelman L, Kim JB, Najib J, Martin G, Fruchart JC, Briggs M, Spiegelman BM, Auwerx J. Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: implications for adipocyte differentiation and metabolism. Mol Cell Biol (1999) 19:5495–5503.[Abstract/Free Full Text]
  30. Hauser S, Adelmant G, Sarraf P, Wright HM, Mueller E, Spiegelman BM. Degradation of the peroxisome proliferator-activated receptor gamma is linked to ligand-dependent activation. J Biol Chem (2000) 275:18527–18533.[Abstract/Free Full Text]
  31. Nicol CJ, Adachi M, Akiyama TE, Gonzalez FJ. PPARgamma in endothelial cells influences high fat diet-induced hypertension. Am J Hypertens (2005) 18:549–556.[CrossRef][Web of Science][Medline]
  32. Ogihara T, Rakugi H, Ikegami H, Mikami H, Masuo K. Enhancement of insulin sensitivity by troglitazone lowers blood pressure in diabetic hypertensives. Am J Hypertens (1995) 8:316–320.[CrossRef][Web of Science][Medline]
  33. Umeji K, Umemoto S, Itoh S, Tanaka M, Kawahara S, Fukai T, Matsuzaki M. Comparative effects of pitavastatin and probucol on oxidative stress, Cu/Zn superoxide dismutase, PPAR-gamma, and aortic stiffness in hypercholesterolemia. Am J Physiol Heart Circ Physiol (2006) 291:H2522–H2532.[Abstract/Free Full Text]
  34. Martin G, Duez H, Blanquart C, Berezowski V, Poulain P, Fruchart JC, Najib-Fruchart J, Glineur C, Staels B. Statin-induced inhibition of the Rho-signaling pathway activates PPARalpha and induces HDL apoA-I. J Clin Invest (2001) 107:1423–1432.[CrossRef][Web of Science][Medline]
  35. Paumelle R, Blanquart C, Briand O, Barbier O, Duhem C, Woerly G, Percevault F, Fruchart JC, Dombrowicz D, Glineur C, Staels B. Acute antiinflammatory properties of statins involve peroxisome proliferator-activated receptor-alpha via inhibition of the protein kinase C signaling pathway. Circ Res (2006) 98:361–369.[Abstract/Free Full Text]
  36. Ray KK, Cannon CP. The potential relevance of the multiple lipid-independent (pleiotropic) effects of statins in the management of acute coronary syndromes. J Am Coll Cardiol (2005) 46:1425–1433.[Abstract/Free Full Text]

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