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Cardiovascular Research 2007 75(2):398-407; doi:10.1016/j.cardiores.2007.03.006
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Copyright © 2007, European Society of Cardiology

Long-term fenofibrate treatment impairs endothelium-dependent dilation to acetylcholine by altering the cyclooxygenase pathway

Javier Blanco-Riveroa,1, Iván Márquez-Rodasb,1, Fabiano E. Xavierc, Rosa Aras-Lópeza, Irene Arroyo-Villaa, Mercedes Ferrera and Gloria Balfagóna,*

aDepartamento de Fisiología, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, 28029 Madrid, Spain
bServicio de Oncología Médica, Hospital General Universitario Gregorio Marañón, Madrid, Spain
cDepartamento de Fisiologia e Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recife, Brazil

* Corresponding author. Tel.: +34 91 4975450; fax: +34 91 4975353. gloria.balfagon{at}uam.es

Received 31 October 2006; revised 16 February 2007; accepted 9 March 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective Experimental studies and opinion articles emphasize that cardiovascular alterations associated with ageing can be improved by the long-term use of fenofibrates. We analyzed the effect of fenofibrate treatment on the acetylcholine-induced relaxation in rat aorta and the participation of nitric oxide (NO) and cyclooxygenase (COX)-derived factors in this effect.

Methods Acetylcholine relaxation in untreated and 6-week fenofibrate-treated Wistar rats was analyzed in the absence and presence of the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the specific inducible NO (iNOS) synthase inhibitor 1400W, the nonspecific COX inhibitor indomethacin, the specific COX-2 inhibitor NS-398, the specific thromboxane receptor antagonist SQ-29548, the thromboxane synthesis inhibitor furegrelate, the prostacyclin synthesis inhibitor tranylcypromine, or the 20-HETES synthesis inhibitor formamidine. eNOS, iNOS, COX-1, and COX-2 expression was studied by Western blotting. In addition, production of prostaglandin F2{alpha} (PGF2{alpha}), thromboxane A2 (TxA2), prostaglandin E2 (PGE2), isoprostanes, and prostacyclin (PGI2) was also measured.

Results Fenofibrate treatment reduced acetylcholine relaxation. Indomethacin, NS-398, and tranylcypromine decreased acetylcholine relaxation in untreated rats but enhanced relaxation in treated rats. SQ-29548 increased acetylcholine responses in segments from treated rats but not in segments from untreated rats. L-NAME decreased vasodilator response to acetylcholine in both groups while furegrelate, NS-398, 1400W, and formamidine did not affect acetylcholine responses in either group. eNOS and COX-2 expression was higher in aorta from treated rats while COX-1 and iNOS remained unmodified. Basal and acetylcholine-stimulated NO and PGE2 release were increased, and that of PGI2 decreased in treated rats. TxA2 release was similar, but PGF2{alpha} release was undetectable in both groups.

Conclusions Although it increases NO production through increases in eNOS expression, fenofibrate treatment induces endothelial dysfunction. This effect seems to be mediated by decreased PGI2 and increased PGE2 release, and it may help to explain the rise in thromboembolic events observed after long-term fenofibrate treatment in humans.

KEYWORDS Fenofibrate; Nitric oxide; Endothelial dysfunction; Prostaglandins


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Endothelium controls the tone of the underlying vascular smooth muscle through the release of vasodilator and contractile factors. These include nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), endothelin-1 and prostaglandins. In general terms, endothelial dysfunction is characterized by reduced endothelium-dependent relaxation, suggesting a reduced availability of NO attributable to either a reduction of NO production and/or enhanced NO inactivation [1,2]. In addition, increased production of cyclooxygenase (COX)-derived prothrombotic and vasoconstrictor factors, including the prostaglandin H2 (PGH2), prostaglandin E2 (PGE2), thromboxane A2 (TxA2) and prostaglandin F2{alpha} (PGF2{alpha}) could also be responsible for the reduced endothelium-dependent relaxation [1].

Fenofibrate, a synthetic ligand of peroxysome proliferator-activated receptor-alpha (PPAR{alpha}) is a useful drug in the treatment of several pathologies associated to dyslipidemia. Experimental studies and expert opinion emphasize that cardiovascular disease prevention can also be improved by strategies that include the long-term use of fenofibrate [3], specially in aging [4]. These beneficial effects of fenofibrate on treatment or prevention of cardiovascular diseases are attributable to its ability to lower triglyceride levels, raise high-density lipoprotein levels, and decrease the levels of low-density lipoprotein [5]. In addition, some studies showed that fenofibrate can improve endothelial function and attenuate the oxidized LDL-induced impairment of endothelial-dependent relaxation [6]. Several works have demonstrated that fenofibrate has inhibitory effects on some thrombotic processes, such as thrombin-induced endothelin-1 production in human endothelial cells [7], serum fibrinogen level [8], red cell aggregation [9] and inhibition of platelet activation [10]. However, an increase in venous thromboembolic events has been observed in the long-term fenofibrate therapy groups in the FIELD study [11].

Recently, it has been suggested that the beneficial effects of fenofibrate on vascular function may be partly due to an improvement in endothelial NO availability, which seems to be mediated by an up-regulation of NO synthase activity and/or expression and by the antioxidant effect of fenofibrate that results in enhanced NO bioactivity [12,13]. Most of the studies about the effects of fenofibrate on the NO pathway have been performed in cell culture and little information is available about the effects of long-term fenofibrate treatment in a living model. On the other hand, NO is crucially involved in the regulation of synthesis and/or release of other vasoactive factors such as arachidonic acid–COX pathway-derived factors, which are implicated in vascular function and thromboembolic events [14]. Taken together, we speculated that long-term treatment with fenofibrate may stimulate NO production and this could affect the production of other endothelial-derived factors as well as the endothelial function.

Therefore, the aim of our study was to assess the long-term effect of fenofibrate treatment on the participation of NO and COX-derived factors on endothelial relaxation induced by acetylcholine. In addition, the production of basal and acetylcholine-stimulated NO and COX-derived prostacyclin (PGI2), PGE2, TxA2 and PGF2{alpha} and 8-isoprostanes was also analyzed.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Animal housing
Male Wistar rats (4 months old) were obtained from colonies maintained at the Animal Quarters of the Facultad de Medicina of the Universidad Autónoma de Madrid. Rats were housed at a constant room temperature, humidity and 12-h light/dark cycle and had free access to tap water and standard rat chow. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

To investigate the response to fenofibrate treatment, oral doses were given according the method described by Soria et al. [15]. Rats were divided into three groups: vehicle and two different fenofibrate treatments (100 mg kg–1 day–1 for either 1 or 6 weeks). Doses used in the current work were selected according to those usually used in chronic experiments. Systolic Blood Pressure was measured in awake animals by the tail-cuff method (Letica, Digital Pressure Meter, LE5000, Barcelona, Spain) before and after the treatment period.

2.2 Vascular reactivity study
Rats were decapitated and the thoracic aorta was removed and placed in cold oxygenated Krebs–Henseleit bicarbonate buffer (KHB). The buffer consisted of (in mM): NaCl 118; KCl 4.7; NaHCO3 25; CaCl2·2H2O 2.5; KH2PO4 1.2; MgSO4·7H2O 1.2; glucose 11 and ethylenediamine-tetraacetic acid 0.01. Segments of thoracic aorta (3 mm in length), free of fat and connective tissue, were mounted between two steel hooks in isolated tissue chambers containing gassed (95% O2 and 5% CO2) KHB, at 37 °C, under a resting tension of 1 g. Isometric tension was recorded by using an isometric force displacement transducer (Grass FTO3C) connected to a 7D Grass polygraph.

After a 45-min equilibration period, each aortic ring was exposed twice to KCl (75 mmol/L), to assess its maximum contractility. After 30 min, rings were contracted with a concentration of noradrenaline that induced approximately 1 g of contraction (noradrenaline-induced contraction: 1.1±0.35 g), and then acetylcholine (0.1 nmol/L to 10 µmol/L) was added to assess the endothelium-dependent relaxation.

To determine the participation of NO in response to acetylcholine, arteries from all experimental groups were incubated with NG-nitro-L-arginine methyl ester (L-NAME, 100 µmol/L, a non-selective NOS inhibitor) or N-(3-aminomethyl) benzylacetamidine (1400W), (1 µmol/L, a selective iNOS inhibitor). These drugs were added 30 min before the concentration–response curve to acetylcholine.

To determine the participation of endothelial vasodilator factors on response to acetylcholine, segments from all experimental groups were incubated with the inhibitors of COX-derived prostanoid and prostacyclin (PGI2) synthesis: indomethacin (10 µmol/L) and tranylcypromine (TCP, 10 µmol/L), respectively. These drugs were added 30 min before the concentration–response curve to acetylcholine. To clarify the participation of COX isoforms on the fenofibrate effects on the acetylcholine-induced relaxation, segments were previously incubated with the specific COX-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398, 10 µmol/L). In another set of experiments, segments were incubated with the thromboxane receptor (TP) antagonist [1S-[1a,2a(Z),3a,4a]]-7-[3-[[2-(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo [2.2.1] hept-2-yl]-5-heptanoic acid, (SQ-29548) (1 µmol/L) to evaluate a possible participation of vasoconstrictor prostanoids. Finally, segments were incubated with the TxA2 synthase inhibitor, furegrelate (1 µmol/L) or the 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthesis inhibitor, N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (0.1 µmol/L) to analyze the possible involvement of these vasoactive factors in the effect of fenofibrate on acetylcholine vasodilation. Relaxation to sodium nitroprusside (0.1 mol/L to 10 µmol/L) or exogenous PGI2 (0.1 nmol/L to 1 µmol/L) was also analyzed in segments pre-contracted with noradrenaline.

2.3 Nitric oxide release
Aortic segments from treated and untreated rats were subjected to a resting tension of 1 g as indicated in the reactivity experiments. After an equilibration period of 60 min, arteries were incubated with the fluorescent probe 4,5-diaminofluorescein (0.5 µmol/L) for 45 min and medium was collected to measure basal NO release. Once the organ bath was refilled, an acetylcholine concentration–response curve (0.1 nmol/L to 10 µmol/L) was applied at 2-min intervals. The fluorescence of the medium was measured at room temperature using a spectrofluorimeter (LS50 Perkin Elmer instruments, FL WINLAB Software) with excitation wavelength set at 495 nm and emission wavelength at 515 nm. The stimulated NO release was calculated by subtracting basal NO release from that evoked by acetylcholine. Also, blank measurement samples were collected in the same way but without aorta segments in order to subtract background emission. Some assays were performed in the presence of L-NAME (100 µmol/L) to ensure the specificity of the method. The amount of NO released was expressed as arbitrary units/mg tissue.

2.4 Western blot analysis for eNOS, iNOS, COX-1 and COX-2
For Western blot analysis of eNOS, iNOS, COX-1 and COX-2 protein expression, aortic segments from untreated and treated rats were homogenized in a boiling buffer composed of 1 mmol/L sodium vanadate (Sigma-Aldrich), 1% SDS and pH 7.4, 0.01 mol/L Tris–HCl, the protein content was measured with a DC protein assay kit (Bio-Rad). Homogenates containing 20 µg protein were electrophoretically separated on a 7.5% SDS–polyacrylamide gel (SDS–PAGE) for eNOS and iNOS, and on a 10% SDS–PAGE for COX-1 and COX-2, then transferred to a polyvinyldifluoride membrane (Bio-Rad). Membranes were blocked in 5% nonfat milk in Tris–HCl buffered saline 0.1% Tween 20 (TBS-T). Subsequent washes were done in TBS-T, and the membranes were incubated with antibody against eNOS and iNOS (1:1000, Transduction Laboratories), COX-1 and COX-2 (1:1000; Cayman Chemical) or {alpha}-actin (1:3000; Sigma-Aldrich) proteins and individual horseradish peroxidase-conjugated secondary antibodies in blocking buffer. Immunoreactive proteins were detected by chemiluminescence with ECL Plus (Amersham). Appropriate positive controls, i.e., human umbilical vein endothelial cells (HUVEC) for eNOS, activated macrophages for iNOS and COX-2, and platelets for COX-1, were used.

2.5 Prostanoid production
The production of TxA2, PGI2, PGE2, PGF2{alpha} and isoprostane is typically monitored by measuring their stable metabolites TxB2, 6-keto-PGF1{alpha}, 13,14-dihydro-15-keto PGF2{alpha}, and 8-isoprostanes, respectively, using the respective Thromboxane B2 EIA Kit, 6-keto-Prostaglandin F1{alpha}, 13,14-dihydro-15-keto Prostaglandin F2{alpha} EIA Kit and 8-Isoprostane EIA Kits (all kits from Cayman Chemical, USA). Segments of rat aorta were pre-incubated for 30 min in 5 mL of KHS at 37 °C and continuously gassed with a 95% O2–5% CO2 mixture (stabilization period). After several 10-min washout periods in a bath of 0.2 mL of KHS, the medium was collected to measure basal prostanoid release. Once the chamber was refilled, the acetylcholine concentration–response curve (0.1 nmol/L to 10 µmol/L) was applied at 2-min intervals. Different assays were made following the manufacturer's instructions. Results were expressed as pg prostanoid mL mg–1 tissue–1.

2.6 Drugs
The drugs used were: L-noradrenaline hydrochloride, acetylcholine chloride, L-NAME hydrochloride, indomethacin, TCP, NS-398, SQ-29548, formamidine and furegrelate. Stock solutions (10 mmol/L) of drugs were made in distilled water; except for noradrenaline, which was dissolved in a NaCl (0.9%)–ascorbic acid (0.01% w/v) solution; indomethacin and formamidine which were solubilized in ethanol and 1400W in methanol and administered from a prepared stock in such a way that the maximal ethanol concentration of the medium was less than 0.001% (vol./vol.); NS-398, which was solubilized in DMSO. All stock solutions were kept at –20 °C, and appropriate dilutions were made in KHS on the day of the experiment.

2.7 Statistical analysis
Results are expressed as mean±S.E.M. for the number of rats indicated. Results of eNOS, iNOS, COX-1 and COX-2 expression are expressed as the ratio between optical density for eNOS, iNOS, COX-1 and COX-2 and for {alpha}-actin. Statistical analysis compared the curve obtained in the presence of the different substances with the control curve by means of repeated-measure two-way analysis of variance (ANOVA). The maximum responses (Emax values) and the logarithms of the acetylcholine concentration producing 20% of maximum response (log EC20) were calculated by a nonlinear regression analysis of each individual concentration–response curve using Graph Pad Prism Software (San Diego, CA). For the experiments on NO and prostanoids release, the statistical analysis employed Student's t test for unpaired experiments. A P value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Blood pressure
In both the 1- and 6-week treatment period, fenofibrate did not modify systolic blood pressure levels in Wistar rats (untreated: 132±4.2 mm Hg; 1 week: 132±6.9 mm Hg; 6 weeks: 139±2.5 mm Hg).

3.2 Vascular reactivity
After 1 week of 100 mg kg day–1 of fenofibrate, vasodilation induced by acetylcholine was comparable to that observed in aortic segments from untreated rats (Fig. 1). However, after the 6-week fenofibrate treatment, the endothelial vasodilator response to acetylcholine was significantly impaired (Fig. 1). In all the experimental groups, the endothelium-independent vasodilation to the NO donor sodium nitroprusside was similar (results not shown). Therefore, since values in control and 1-week treated rats were similar, the rest of this study will be performed in rats treated with 100 mg kg day–1 of fenofibrate for 6 weeks.


Figure 1
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  		Fig. 1 Effect of 1- and 6-week fenofibrate treatment on the concentration-dependent relaxation to acetylcholine in aortic segments from Wistar rats. Results (mean±S.E.M.) are expressed as percent of the previous contraction to noradrenaline.

 
Pre-incubation with the non-selective NOS inhibitor L-NAME diminished the vasodilator response to acetylcholine to a similar level in segments from treated and untreated rats (Fig. 2A and B). However, in presence of the selective iNOS inhibitor 1400W, relaxation to acetylcholine remained unmodified in both groups (Fig. 2A and B).


Figure 2
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  		Fig. 2 Effect of L-NAME or 1400W on the concentration-dependent relaxation to acetylcholine in aortic segments from untreated (A) and 6-week fenofibrate-treated (B) rats. Results (mean±S.E.M.) are expressed as percent of the previous contraction to noradrenaline.

 
Pre-incubation of aortic segments from untreated rats with either indomethacin or the specific PGI2 synthesis inhibitor TCP diminished the endothelial vasodilator response to acetylcholine (Fig. 3A). However, in segments from fenofibrate-treated rats this vasodilator response was enhanced in the presence of indomethacin and remained unmodified in the presence of TCP (Fig. 3B). The vasodilator response to exogenous PGI2 was not modified by fenofibrate treatment (results not shown). Incubation with the specific COX-2 inhibitor NS-398 decreased the acetylcholine-induced relaxation in segments from the untreated group but increased it in segments from the fenofibrate-treated rats (Fig. 3A and B). SQ-29548 also increased the acetylcholine-induced relaxations in treated rats, but did not modify this response in arteries from untreated rats (Fig. 4A and B). Incubation with the thromboxane synthase inhibitor furegrelate or the 20-HETE synthesis inhibitor formamidine did not modify endothelium-dependent relaxation in aorta segments from either treated or untreated animals (Fig. 4A and B).


Figure 3
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  		Fig. 3 Effect of indomethacin, TCP or NS-398 on the concentration-dependent relaxation to acetylcholine in aortic segments from untreated (A) and 6-week fenofibrate-treated (B) rats. Results (mean±S.E.M.) are expressed as percent of the previous contraction to noradrenaline.

 

Figure 4
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  		Fig. 4 Effect SQ-29548, furegrelate or formamidine on the concentration-dependent relaxation to acetylcholine in aortic segments from untreated (A) and 6-week fenofibrate-treated (B) rats. Results (mean±S.E.M.) are expressed as percent of a previous contraction to noradrenaline.

 
The effect of different drugs used on the maximum response to acetylcholine (Emax) and EC20 are summarized in Table 1.


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  		Table 1 Changes in the maximum response (Emax, expressed as a percentage of relaxation) and log EC20 to acetylcholine of intact thoracic aorta from untreated and fenofibrate-treated rats

 
3.3 Nitric oxide release
Basal and stimulated NO release in response to acetylcholine was enhanced in aortic segments from fenofibrate-treated rats compared to untreated rats (Fig. 5).


Figure 5
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  		Fig. 5 Effect of fenofibrate treatment on the basal and acetylcholine-induced NO release in aorta from Wistar rats; means±S.E.M. *P<0.05 acetylcholine-stimulated vs. basal; #P<0.05 fenofibrate-treated vs. untreated rats.

 
3.4 eNOS, iNOS, COX-1 and COX-2 protein expression
eNOS and COX-2 protein expression was higher in aortic segments from fenofibrate-treated rats compared to untreated animals while iNOS and COX-1 expression were similar (Fig. 6).


Figure 6
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  		Fig. 6 Representative Western blot for eNOS and COX-2 (A) and iNOS and COX-1 (B) expression in aortic segments from untreated and fenofibrate-treated rats. Figure is representative of four separate experiments. Panels C and D show densitometric analysis of the Western blot for COX-2 and eNOS protein expression, respectively. Results (means±S.E.M.) are expressed as the ratio between the signal for the eNOS and COX-2 protein and the {alpha}-actin signal. *P<0.05 fenofibrate-treated vs. untreated rats.

 
3.5 Prostanoid production
Treatment with 100 mg kg–1 day–1 of fenofibrate during 6 weeks did not modify basal or acetylcholine-stimulated TxB2 and 8-isoprostane levels (Fig. 7A and B). The 6-keto-PGF1{alpha} levels were decreased compared to untreated rats, while PGE2 levels were increased (Fig. 7C and D). The levels of 13,14-dihydro-15-keto PGF2{alpha} were not detectable in either treated or untreated rats.


Figure 7
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  		Fig. 7 Effect of fenofibrate treatment on the basal and acetylcholine-stimulated production of TxB2 (A), isoprostanes (B), 6-keto-PGF1{alpha} (C) and PGE2 (D) in aortic segments from untreated and fenofibrate-treated rats. *P<0.05 fenofibrate-treated vs. untreated rats. #P<0.05 fenofibrate-treated vs. untreated rats.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Fenofibrate is a synthetic ligand for PPAR{alpha}, which is known mainly for its lipid-lowering effect [5]. Furthermore, apart from that effect, fenofibrate has been considered a vascular anti-inflammatory and anti-thromboembolic drug through its decrease of plasma inflammatory cytokines and thromboembolic products, respectively [16,17]. Clinical trials of fenofibrate in patients with diabetes type 2 and with dyslipidemia report an improvement in impaired cardiovascular parameters associated to an improvement of endothelial function [18,19]. This beneficial effect of fenofibrate on endothelial function seems to be mediated by an increase of endothelial NO release and/or bioavailability related to the inhibition of inflammatory pathways in the arterial wall, the antioxidant effect of fenofibrate and the enhancement of endothelial NOS (eNOS) expression and activity [13,16]. Endothelium-derived NO is an important mediator of cardiovascular protection through its regulatory effects on vascular tone, platelet aggregation, oxidative stress, leukocyte adherence and vascular smooth muscle cell proliferation [20]. Additionally, NO is crucially involved in the regulation of synthesis and/ or release of other vasoactive factors such as arachidonic acid–COX pathway-derived factors [14]. The above-mentioned results led us to speculate that the enhanced NO production induced by fenofibrate might affect the release of other endothelial-derived factors and thereby affect endothelial function. The current study was designed to compare the effect of long-term fenofibrate treatment on the endothelial dilation induced by acetylcholine and the contribution of NO and COX-derived products to this response.

Surprisingly, after 6 weeks of fenofibrate treatment, the endothelium-dependent vasodilation induced by acetylcholine was reduced in isolated rat aortic rings, but had remained unaltered after the 1-week treatment, indicating that fenofibrate induces endothelial dysfunction in Wistar rats in a time-dependent manner. It is important to note that the dose used in the current study is comparable to that usually employed in experimental studies [6,21,22]. This deleterious effect induced by fenofibrate on endothelial function does not seem to be mediated by hemodynamic changes, since no differences in arterial pressure were observed between treated and untreated animals, in contrast to reports in humans [23].

Endothelial dysfunction is usually associated with a reduction in NO production and/or increases in NO metabolism [1,2]. In regard to the effects of fenofibrate on the endothelial NO pathway, it has previously been reported that fenofibrate increases eNOS expression and/or its activity in cultured endothelial cells [13] suggesting that this direct effect, leading to increases in NO production, could contribute to the improvement of endothelial function. In the current study, we observed that in aortic segments from both experimental groups, L-NAME, but not 1400W, reduced the acetylcholine-induced relaxation to a similar level, although acetylcholine-induced NO release and eNOS expression were increased in arteries from fenofibrate-treated rats. Taking into account that the vasodilator response to the NO donor sodium nitroprusside was not altered by fenofibrate treatment, our results suggest that, although NO production is enhanced by fenofibrate treatment, its contribution to acetylcholine-induced relaxation seems to be reduced. Considering that endothelial cells also release other vasoactive factors when stimulated by acetylcholine [24], we think that the decreased endothelium-dependent vasodilation in arteries from fenofibrate-treated rats could be mediated by a decrease and/or increment in endothelial production of vasodilating and/or vasoconstricting factors, respectively.

The biosynthesis and release of NO and prostaglandins share a number of similarities. The enhanced release of prostaglandins is partly driven by NO, suggesting that COX enzymes would be important actors in the modulation of NO effects [14]. We have observed increased NO production in fenofibrate-treated rats and, since COX-derived products play an important role in endothelial dysfunction [25], we analysed the possible participation of COX derivates in altered acetylcholine dilation. Participation was confirmed by the normalization of acetylcholine relaxation in aortic segments of fenofibrate-treated rats in the presence of the COX-1/COX-2 inhibitor indomethacin. It is important to note that indomethacin decreased the acetylcholine responses in segments from untreated rats but enhance it in segments from fenofibrate-treated rats. These results suggest that in segments from untreated rats, the COX pathway contributes to the acetylcholine response with a vasodilator effect while in fenofibrate-treated animals the effect of COX pathway is predominantly vasoconstrictor. The present results obtained in the presence of the PGI2 synthesis inhibitor TCP indicate that PGI2 plays a greater role in the vasodilator response induced by acetylcholine in aortic segments from untreated animals than in the fenofibrate-treated animals. Corroborating these results is the fact that PGI2 release is decreased in segments from fenofibrate-treated animals while the vasodilator response to exogenous PGI2 remains unmodified. These results indicate that the decreased PGI2 release could play a role in the decreased vasodilator response to acetylcholine induced by the 6-week fenofibrate treatment.

As indicated by the results observed in the presence of indomethacin, increased vasoconstrictor prostanoid release might potentially be a mechanism also implicated in the endothelial dysfunction induced by fenofibrate treatment. It has been reported that through the activation of TP receptors, vasoconstrictor prostanoids such as PGH2, PGE2, TxA2 and PGF2{alpha} can participate in the endothelial dysfunction associated with different cardiovascular risk factors [26,27]. Incubation with SQ-29548, a TP receptor antagonist, did not modify the acetylcholine-induced relaxation in segments from untreated rats but increased it in fenofibrate-treated rats. This result suggests the participation of vasoconstrictor prostanoids in endothelial dysfunction induced by fenofibrate treatment. In both groups, acetylcholine increased TxA2 release to the same extent while PGF2{alpha} release was undetectable in both experimental groups. Acetylcholine induced PGE2 release in aortic segments from both groups, but in a greater extent in segments from fenofibrate-treated animals. Additionally, in both experimental groups, incubation with furegrelate did not modify the vasodilator response to acetylcholine. Consequently TxA2 and PGF2{alpha} do not seem to participate in the impaired acetylcholine relaxation induced by fenofibrate and this suggests the participation of the vasoconstrictor COX-related compound PGE2. This vasoconstrictor product is possibly derived from the inducible COX isoform since the specific COX-2 inhibitor NS-298 also increased the vasodilation to acetylcholine and COX-2 expression in segments from fenofibrate-treated rats. Reinforcing this hypothesis, our results showed that COX-1 expression was not altered by fenofibrate treatment.

20-HETE is endogenously produced by {omega}-hydroxylases of the cytochrome P450 in vascular wall. This increases smooth muscle tone indirectly as a result of its metabolism by COX in endothelial cells, inasmuch as indomethacin and SQ-29548 inhibit endothelium-dependent contraction [28]. We analysed the possibility that 20-HETE derivates could participate in the endothelial dysfunction induced by fenofibrate treatment. Incubation with formamidine did not modify the relaxation induced by acetylcholine in segments from treated and untreated rats, discounting the participation of 20-HETE derivates in this response.

Isoprostanes are a family of compounds produced from arachidonic acid via a free-radical catalyzed mechanism. These compounds induce a potent vasoconstriction, primarily mediated by TP receptor stimulation and, in some vessels, by the release of COX products [29]. Fenofibrate treatment was unable to increase the isoprostanes release induced by acetylcholine, ruling out its contribution to the effect of fenofibrate.

Although the majority of works published refers the PPARs as anti-inflammatory, some studies indicate that PPAR{alpha} and PPAR{gamma} could have pro-inflammatory effect [30,31]. Based on these previous studies and the results presented here it is important to consider its possible implications for the therapeutic use of PPAR{alpha} agonists in some pathologies and preventive treatments. This pro-inflammatory effect could help to explain the rise in thromboembolic events observed after long-term fenofibrate treatment in humans [11]. However, further investigations using other animal models are necessary to further understand the underlying mechanisms of fenofibrate-induced endothelial dysfunction before making definite conclusions.

In conclusion, although fenofibrate treatment increases NO production through increases in eNOS expression, this drug induces endothelial dysfunction in a time-dependent manner in aortic segments from Wistar rats. This effect seems to be mediated by a decrease in PGI2 release and an increase in PGE2 production.

Time for primary review 19 days


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This research was supported by the Fondo de Investigaciones Sanitarias (PI051767), the Comisión de Ciencia y Tecnología (SAF 2005-05760 and SAF-2006-07888) and Banco de Santander-UAM. We are grateful to Dr. M. C. Fernández-Criado for care of the animals.


   Notes
 
1 Should be considered as first authors. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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