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Title Page

Endothelium-dependent vasodilator effects of PPAR β agonists via the PI3K-Akt

pathway

Rosario Jiménez, Manuel Sánchez, María José Zarzuelo, Miguel Romero, Ana María

Quintela, Rocío López-Sepúlveda, Pilar Galindo, Manuel Gómez-Guzmán, Jose

Manuel Haro, Antonio Zarzuelo, Francisco Pérez-Vizcaíno and Juan Duarte

Department of Pharmacology, School of Pharmacy, University of Granada (RJ, MS, MJZ,

MR, RLS, PG, MGG, AZ and JD) and Ciber Enfermedades Hepáticas y Digestivas

(CIBEREHD) (AZ), Service of Gynecology, Clinic Hospital of Granada, Granada (JMH)

and Department of Pharmacology, School of Medicine, University Complutense of Madrid

and Ciber Enfermedades Respiratorias (CIBERES) (FPV), Madrid, Spain.

JPET Fast Forward. Published on November 11, 2009 as DOI:10.1124/jpet.109.159806

Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.

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Running Title Page: Vasodilator effects of PPAR-β agonists

Correspondence to: Juan Duarte. Department of Pharmacology, School of Pharmacy.

University of Granada, Spain.Tel: (34)-958244088; Fax. (34)-958248264. Email

address: jmduarte@ugr.es

Number of text pages: 28

Number of tables: 0

Number of figures: 6

Number of references: 42

Number of words in the abstract: 240

Number of words in the introduction: 546

Number of words in the discussion: 1345

List of nonstandard abbreviations:

Akt, protein kinase B; DMSO, dimethylsulfoxide; eNOS, endothelial nitric oxide

synthase; HDL, high-density lipoprotein; HUVECs, human umbilical vein endothelial

cells; NO, nitric oxide; PBS, physiological buffer saline; Phe, phenylephrine; PI3K,

phosphatidyl-inositol-3 kinase; PPARs, Peroxisome Proliferator-Activated Receptors;

TBST, Tris-buffered saline (containing 0.1 % Tween 20); VSMC, vascular smooth

muscle cell

Recommended section: Cardiovascular

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Abstract

Peroxisome proliferator-activated receptor β/δ (PPAR-β) is a ligand-activated transcription

factor belonging to the nuclear hormone receptor superfamily that regulates the

transcription of many target genes. Recently, acute, non-genomic effects of PPAR β

agonists have also been described. In the present study we hypothesized that PPAR β

agonists might exert acute non-genomic effects on vascular tone. Here we report that the

structurally unrelated PPAR-β ligands, L165041 and GW0742, induced vascular relaxation

in phenylephrine-pre-contracted endothelium intact rat aortic rings, which was

significantly inhibited by endothelial denudation or nitric oxide synthase (NOS) inhibition

with NG-Nitro-L-Arginine methylester. These relaxant effects reached steady-state within

15 min. The relaxation induced by both L165041 and GW0742, in aortic rings pre-

contracted with the thromboxane A2 analogue U-46619 was unaffected either by removal

of extracellular calcium or by incubation with calcium-free solution containing the

intracellular calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N, N, N’, N’-tetraacetic

acid tetra(acetoxymethyl) ester. However, the phosphatidyl-inositol-3 kinase (PI3K)

inhibitor, LY-294002, inhibited the endothelium-dependent relaxant responses induced by

both PPAR-β agonists. Blockade of PPAR-β with GSK0660 also partially inhibited these

relaxant responses, being without effect PPARγ blockade with GW9662. In human

umbilical vein endothelial cells, L165041 and GW0742 increased nitric oxide (NO)

production and Akt and eNOS phosphorylation, which were sensitive to PI3K inhibition

and PPAR-β blockade. In conclusion, the PPAR-β agonists, acutely caused vasodilatation

which was partially dependent on endothelial-derived NO. Endothelial NOS activation is

calcium independent and seems to be related to activation of the PI3K-Akt-eNOS pathway.

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Introduction

Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated nuclear

receptors which heterodimerize with the retinoid X receptor to regulate the transcription of

diverse genes (Kota et al., 2005). There are three known PPAR subtypes: α, β (also

referred to as δ), and γ. PPAR-α, found in liver, heart, kidney, skeletal muscle, brown

adipose tissue and vascular and immune cells, is involved mainly in lipid metabolism and

is activated by fibrates. PPAR-γ, expressed principally in adipose tissue, liver, vascular and

immune cells, is related with adipogenesis and glucose homeostasis, and is activated by

thiazolidinediones. Despite PPAR-β is the most widely expressed PPAR receptor in body

tissues, its physiological and pathophysiological role is less known. It has been implied in

adipose tissue formation (Vosper et al., 2001), brain development (Cimini et al., 2005), cell

proliferation (Piqueras et al., 2007), placental function (Barak et al., 2002) and

inflammation (Lee et al., 2003).

PPAR-α and/or PPAR-γ ligands are widely used in the treatment of dyslipidemia

and type-2 diabetes mellitus, respectively. Beyond their metabolic effects on blood glucose

and lipids, they show a favourable cardiovascular profile due to their well known

antiatherosclerotic, anti-inflammatory and vasodilator effects (Schiffrin et al., 2003), and

their abilities to inhibit endothelial and vascular smooth muscle cell (VSMC) proliferation

(Lee et al., 2006; Benkirane et al., 2006), to reduce cardiac hypertrophy (Asakawa et al.,

2002), to inhibit platelet aggregation (Moraes et al., 2007) and to decrease blood pressure

(Khan et al., 2005; Iglarz et al., 2003). PPAR-β ligands have been more recently developed

and are currently in clinical trials for the treatment of dyslipidemia (Risérus et al., 2008;

Barish et al., 2006). However, less is known about other non-metabolic effects of these

drugs. So far, it has been demonstrated that PPAR-β activation improves cardiac

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hypertrophy in vitro (Sheng et al., 2008), protects human umbilical vein endothelial cells

(HUVECs) from hydrogen peroxide-induced apoptosis (Jiang et al., 2009) and inhibits

VSMC proliferation and migration (Lim et al., 2009) but, paradoxically, it induces

endothelial cell proliferation and angiogenesis (Piqueras et al., 2007; Han et al., 2008).

Recently, multiple non-genomic effects have been reported for agonists of different

nuclear receptors (Burgermeister and Seger, 2007; Cheskis et al., 2007; Stahn and

Buttgereit, 2008). Thus, PPAR-γ ligands inhibit the nuclear factor-κ B (Chen et al., 2003)

or retinoid X receptor ligands exert antiagregant effects (Moraes et al., 2007). PPAR-β

agonists also inhibit proliferation and migration of rat VSMC (Han et al., 2008) and inhibit

platelet aggregation and activation (Ali et al., 2006) via non genomic effects. We

hypothesized that PPAR-β agonists could exert vasodilatory effects via non genomic

mechanisms.

Due to the promising therapeutic role of PPAR-β agonists and the relative lack of

knowledge of the actions of PPAR-β in the vascular territory, in this study we investigated

the short-term effects of [4-[3-(4-Acetyl-3-hydroxy-2 propylphenoxy)propoxy]

phenoxy]acetic acid (L-165041), a weak nonselective PPAR-β agonist (Willson et al.,

2000), and 4-[[[2-[3-Fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5 -thiazolyl]methyl]thio]-

2-methylphenoxy]acetic acid (GW0742), a selective PPAR-β agonist (Kim et al., 2006), on

vascular tone in isolated rat aortic rings, focusing in the role of endothelium and nitric

oxide (NO), and their effects on the expression and phosphorylation of endothelial nitric

oxide synthase (eNOS) in HUVEC. A preliminary account of these results was presented at

the 2007 Winter Meeting of the British Pharmacological Society (Jimenez et al., 2007).

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Methods

Tissue preparation and measurement of tension

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) and with the principles outlined in the Declaration of Helsinki and approved by our

institutional review board. Male Wistar rats (250-300 g) were euthanized by a quick blow

on the head followed by exsanguination by trained personnel. The descending thoracic

aortic rings were dissected and mounted in organ chambers filled with Krebs solution

(composition in mM: NaCl, 118; KCl, 4.75; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2; KH2PO4,

1.2; and glucose, 11) at 37°C and gassed with 95% O2 and 5% CO2. Rings were stretched

to 2 g of resting tension by means of two L-shaped stainless-steel wires inserted into the

lumen and attached to the chamber and to an isometric force–displacement transducer

(Letigraph 2000, Letica S.A., Barcelona, Spain) respectively as described previously

(Jiménez et al., 2007), and equilibrated for 60-90 min. In some arteries the endothelium

was mechanically removed by gently rubbing the intimal surface of the rings with a metal

rod. The absence of endothelium was confirmed by the absence of relaxing effects of

acetylcholine (10-6 M) in aortic rings previously contracted by 10-7 M phenylephrine (Phe).

For the experiments in which Ca2+-free Krebs solution was used, CaCl2 was omitted and

0.5 mM ethylene glycol-bis(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA) was

added.

After equilibration, rings with or without endothelium were stimulated by a single

concentration of Phe (titrated to produce the 80% of the maximal contractile response of

the agonist as determined in preliminary experiments) so that a similar tone was achieved

in all experimental conditions. When contractions were stable, concentration–relaxant

response curves were carried out by cumulative addition of the PPAR-β agonists L165041

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or GW0742 (0.1 – 30 μM) at 15 minutes intervals. Addition of vehicle (dimethylsulfoxide,

DMSO, 0.1%) had no significant relaxant effect (2 ± 3 % relaxation at the highest

concentration of DMSO tested, n = 6). In some endothelium-intact aortic rings, the same

protocol was performed in the presence of the eNOS inhibitor NG-nitro-L-arginine

methylester (L-NAME, 10-4 M) for 20 min. To explore if PPAR-β agonists increased the

sensitivity of the NO-cGMP pathway in the vascular smooth muscle, we examined the

relaxant response induced by the NO donor sodium nitroprusside (10-10 -10-6 M), in the

absence or in the presence of L165041 or GW0742 (10 μM), in endothelium-denuded

aortic rings previously contracted with Phe.

To examine the involvement of Ca2+ on the endothelium-dependent relaxation

induced by the PPAR-β agonists, two experimental protocols were performed: a) To test

the role of extracellular Ca2+, intact aortic segments were incubated in Ca2+-free Krebs

solution for 30 minutes prior to the addition of 9,11-dideoxy-11α,9α-

epoxymethanoprostaglandin F2α (U-46619, 0.1 μM), a Ca2+-independent vasoconstrictor

agent, and then a concentration-response curve to L165041 or GW0742 (0.1-30 μM) was

constructed; b) To test the role of intracellular Ca2+, the relaxant responses induced by

these agents were analysed in aortic rings incubated for 30 min with the intracellular

calcium quelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetra

(acetoxymethyl) ester (BAPTA/AM) (10-5 M) and, after washing for 15 min, precontracted

with U-46619 (10 μM) in Ca2+-free-Krebs solution.

To evaluate the involvement of phosphatidyl-inositol-3 kinase (PI3K) in the relaxant

effects of the PPAR-β agonists some aortic rings with endothelium were incubated for 30

min in Krebs solution containing the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-

benzopyran-4-one hydrochloride (LY-294002, 1 μM). Then the vessels were exposed to

Phe (1 μM) and, when the contractile response was stable, a concentration-response curve

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to L165041 or GW0742 was constructed. To demonstrate if these relaxant effects were

related to PPAR-β or to PPAR-γ activation, the effects of L165041 or GW0742 in Phe pre-

contracted rings in the presence of the PPAR-β antagonist 3-[[[2-methoxy-4-

(phenylamino)phenyl]amino]sulfonyl]-2- thiophenecarboxylic acid methyl ester

(GSK0660, 1 μM) or the PPAR-γ antagonist 2-chloro-5-nitro-N-phenylbenzamide

(GW9662, 1 μM) were investigated.

Protein phosphorylation in HUVECs

HUVECs were extracted from umbilical cords (modified from Vargas et al., 1994).

Briefly, HUVECs were isolated by filling the lumen of fresh umbilical veins with 0.1%

collagenase in physiological buffer saline (PBS), inverting the umbilical cord and washing

the vein with culture medium (Medium 199 + 20% fetal bovine serum +

Penicillin/Streptomycin 2mM + Amphotericin B 2 mM + Glutamine 2 mM + HEPES 10

mM + endothelial cell growth supplement 30 μg/ml + Heparin 100 mg/ml). The collected

cells were seeded in culture flasks pre-treated with gelatin 0.2%, containing culture

medium. To perform the western blots, cells were washed and incubated 3 hours only with

medium. Then they were incubated with L165041 or GW0742 (10 μM) for 15 min, and

some with LY-294002 (1 μM), or PPAR-β antagonist GSK0660 (1 μM) or GW9662 (1

μM), 30 min prior and during the PPAR-β agonist exposure. After this period, cells were

washed with PBS and homogenized. Western blots were performed with 30 μg of protein

per lane, previously determined by the bicinchoninic acid assay (Walker et al., 1994).

Sodium dodecyl sulphate-polyacrilamide (8%) electrophoresis was performed in a mini-gel

system (Bio-Rad). The proteins were transferred to polyvinylidene difluoride membranes

for 1 hour which were then blocked with Tris-buffered saline (containing 0.1 % Tween 20)

(TBST) containing 5% non fat dry milk for 90 min at room temperature. Phosphorylated

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protein kinase B (Akt) (Ser-473), phosphorylated eNOS (Ser-1177), Akt and eNOS, were

detected after the membranes were incubated with the respective primary antibodies (rabbit

anti-p-eNOS-ser-1177, mouse anti-eNOS, rabbit anti-p-Akt-ser-473 and rabbit anti-Akt,

1:1000 dilution) overnight at 4ºC. The membranes were then washed three times for 10 min in

TBST and incubated with secondary peroxidase conjugated goat anti-rabbit or goat anti-

mouse antibodies (1:2500), respectively. All incubations were performed at room temperature

for 2 hours. After washing the membranes, antibody binding was detected by an

electrochemiluminescent system. Films were scanned and densitometric analysis was

performed on the scanned images using Scion Image-Release Beta 4.02 software

(http://www.scioncorp.com). Phospho-Akt/Akt and phospho-eNOS/eNOS abundance ratio

was calculated and data is expressed as a percentage of the values in control cells from the

same gel.

Quantification of nitric oxide released by diaminofluorescein-2 in HUVECs

Quantification of NO released by HUVEC was performed using the NO-sensitive

fluorescent probe diaminofluorescein-2 (DAF-2) as described previously (Leikert et al.,

2001). Briefly, cells were grown to confluence in 96-well plates and heparin and

endothelial cell growth supplement were omitted 24 h before stimulation. Cells were

washed with PBS and then were pre-incubated with L-arginine (100 μM in PBS, 5 min,

37ºC). In some experiments, L-NAME (10-4 M) was added 15 min before the addition of

L-arginine. Subsequently, DAF-2 (0.1 μM) and either the calcium inophore calimycin

(A23187, 1 μM) or the PPAR-β agonist L165041 (1, 10 and 30 μM) or GW0742 (1, 10 and

30 μM) were added and cells were incubated in the dark at 37ºC. Then the fluorescence

was measured at 5, 15 and 30 min, respectively, using a spectrofluorimeter (Fluorostart,

BMG Labtechnologies, Offenburg, Germany) with excitation wavelength set at 495 nm

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and emission wavelength at 515 nm. The auto-fluorescence obtained from PBS/DAF-2 was

subtracted from each value. In some experiments, the fluorescence signal induced by

L165041 or GW0742 (30 μM) was measured in HUVECs pretreated for 30 min with LY-

294002 (1 μM), GSK0660 (1 μM), or GW9662 (1 μM).

Materials

L165041, GW0742 and GSK0660 were purchased from Tocris bioscience (Bristol, UK).

Medium 199 and HEPES were obtained from Lonza (Verviers, Belgium). Rabbit anti-p-

eNOS-ser-1177, mouse anti-eNOS, rabbit anti-p-Akt-ser-473 and rabbit anti-Akt were from

Cell Signalling Technology, MA, USA. Secondary peroxidase conjugated goat anti-rabbit and

goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,

USA). Electrochemiluminescent system was from Amersham Pharmacia Biotech

(Amersham, UK). The rest of products were obtained from Sigma Aldrich Chemie

(Steinheim, Germany).

Statistical analysis

Results are expressed as means ± S.E.M. and n reflects the number of experiments

performed. Statistically significant differences between groups were calculated by an

analysis of variance (ANOVA) followed by a Newman-Keuls test. P < 0.05 was

considered statistically significant.

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Results

Vasorelaxant effects of PPAR-β agonists

The addition of Phe to the organ chamber produced a sustained vasoconstriction in isolated

vessels (1600 ± 153 mg). The subsequent addition of L-165041 or GW0742 at the

therapeutic concentrations (ie. μM range, Takata et al., 2008), caused a concentration-

dependent relaxation which reached steady-state within 15 min (Figure 1A). The

contractile response in parallel time controls remained fairly constant (less than 10% decay

after 90 min). The mechanical removal of the endothelium significantly reduced the

vasodilator effect of both agonists. Moreover, the eNOS inhibitor L-NAME abolished the

relaxation induced by both agonists, what suggests that this response involves endothelial-

derived NO (Figure 1B, 1C). Neither L-165041 (10 μM) nor GW0742 (10 μM) modified

the endothelium-independent relaxant response induced by sodium nitroprusside in Phe-

precontracted rings (-log [Inhibitory Concentration 50] = 8.74 ± 0.33, 8.91 ± 0.22, 8.82 ±

0.33, n =5-7, control, L-165041 and GW0742 pretreated rings, respectively).

In endothelium-denuded aortic rings, pre-treatment with L-165041 or GW0742 at

30 μM also inhibited the contractile responses induced by Phe (10 nM - 1 μM) or by

U46619 (10 - 100 nM) (not shown). Moreover, L-165041 and GW0742 (at ≥ 10 μM) also

inhibited the transient (phasic) contraction induced by Phe in the absence of extracelullar

calcium (60 ± 7 and 25 ± 7%, respectively, at 30 μM).

Role of PPAR-β and PPAR-γ in vasorelaxant effects of PPAR-β agonists

The presence of the PPAR-γ antagonist GW9662, did not modify the relaxant responses

induced by both L-165041 and GW0742 in intact arteries precontracted with Phe (Figure

2A and 2B). However, the PPAR-β antagonist, GSK0660, significantly inhibited these

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relaxant effects in arteries with intact-endothelium (Figure 2A and 2B), being without

effects in endothelium-denuded aortic rings (Figure 2C and 2D). None of these inhibitors

modified the concentration-response curve for the relaxations induced by acetylcholine or

sodium nitroprusside (supplemental Figure 1).

Involvement of calcium in endothelium-dependent vasorelaxant effects of PPAR-β

agonists

PPAR-β agonists also relaxed endothelium-intact aortic rings precontracted with the

thromboxane A2 analog U46619 (0.1 μM). These relaxant effects were also inhibited by

vascular endothelium denudation. L165041- or GW0742-induced endothelium-dependent

relaxation of U46619-precontracted rings was not affected by removal of extracellular

Ca2+. Moreover, the incubation of aortic rings in Ca2+-free Krebs solution containing the

intracellular Ca2+ quelator BAPTA-AM did not modify significantly the endothelium-

dependent relaxations induced by both agonists (Figure 3).

Role of PI3-kinase in endothelium-dependent vasorelaxant effects of PPAR-β agonists

We then explored potential Ca2+-independent mechanisms for eNOS activation such as the

PI3K pathway. In the presence of the PI3K inhibitor LY-294002, the relaxant responses

induced by both L-165041 or GW0742 in arteries precontracted with Phe were

significantly inhibited (Figure 4)

Effects of PPAR-β agonists on nitric oxide production in HUVECs

Exposure of HUVECs to the calcium ionophore A23187 (1 μM) increased significantly the

DAF-2 fluorescence intensity as compared with control cells (Figure 5A). This increase

was unaffected by the PI3K inhibitor LY-294002 (191 ± 16 and 224 ± 24 arbitrary units, n

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= 8, measured at 30 min in the presence or absence of the drug, respectively). Similarly, L-

165041 or GW0742, in a concentration- and time-dependent manner, increased L-NAME-

sensitive NO production in HUVECs (Figure 5B, 5C). This NO production, measured at 30

min, was partially reduced by both the PI3K inhibitor LY-294002 and the PPAR-β

antagonist GSK0660, and was unaffected by the PPAR-γ antagonist GW9662 (Figure 5D,

5E).

Effects of PPAR-β agonists in Akt and eNOS phosphorylation in HUVECs

Incubation of HUVECs for 15 min with L-165041 or GW0742 produced an increase in

both Akt (Figure 6A, 6B) and eNOS (Figure 6C, 6D) phosphorylation. Akt and eNOS

phosphorylation was inhibited in presence of the PI3K inhibitor LY-294002 or the PPAR-β

antagonist GSK0660, but was unaffected by the presence of the PPAR-γ antagonist

GW9662.

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Discussion

In our study, we found for the first time, that PPAR-β agonists induced relaxant responses

in isolated rat aortic rings, which were partially dependent on endothelium and NO. The

endothelium-dependent relaxations induced by these agents were calcium-independent and

were sensitive to PI3K inhibition and PPAR-β blockade. Moreover, these agonists induced

NO production and PI3K-dependent Akt and eNOS phosphorylation in cultured HUVECs.

L-165041 and GW0742 are specific PPAR-β agonists (Berger et al., 1999;

Sznaidman et al., 2003). In the present study, these drugs were used in the expected range

of therapeutic concentrations (i.e. micromolar, Takata et al., 2005) at which they are

effective activators of PPAR-β. Both drugs produced a relaxant effect with a similar

pharmacological profile and via the same mechanism of action. Importantly, they belong to

different chemical classes. Moreover, blockade of PPAR-β with the PPAR-β antagonist

GSK0660 also inhibited both the relaxant-response and the endothelial NO production

induced by both PPAR-β agonists. Altogether these data support that the effects of L-

165041 are GW0742 are due to activation of PPAR-β. Concerns may be raised because the

concentrations used herein are 100-1000 times higher than the reported Ki values for

binding PPAR-β (Berger et al., 1999; Sznaidman et al., 2003). However, concentrations of

1 μM or higher are required to observe PPAR-β-dependent effects (either genomic or non

genomic) as used in the vast majority of the studies published with these two drugs.

Further studies are required to analyze the reasons for this discrepancy and/or to identify

other potential receptors or receptor subtypes involved. Moreover, these effects seem to be

independent of PPAR-γ activation, since the PPAR-γ antagonist GW9662 was unable to

inhibit them. The PPAR-α ligand clofibrate had no vasorelaxant effect at concentrations up

to 100 μM (authors unpublished) suggesting that the effects of PPAR-β agonist are

independent of PPAR-α. On the other hand, blockade of PPAR-β did not abolish the

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endothelium-independent vascular responses observed at higher concentrations of the

drugs, which suggests the involvement of PPAR-β-independent pathways.

It has been recently described that PPAR-β localization is not restricted to the

nucleus (Kelly et al., 2004). In addition, it is known that these receptors are present in

VSMC as well as in endothelial cells (Kliewer et al., 1994; Piqueras et al., 2009). We

found that the maximal vasodilator response to every agonist concentration was reached

within 15 minutes, what suggests that it is independent of nuclear processes, because it is

not enough time for gene transcription. These results show for the first time a fast, non-

genomic effect of the PPAR-β agonists in the vascular bed. Ali et al., 2006 have described

the existence of these receptors in non-nucleated cells (platelets), and that their rapid

activation by agonists (5 min) produces inhibition of platelet aggregation.

The vascular endothelium plays an important role in controlling vascular tone via

the release of relaxant and contractile factors (Vanhoutte et al., 1986). It is known that NO

is the most important vasodilator released by the endothelium and, in some vessels such as

rat aorta, the endothelium-dependent vasodilation is almost completely due to the release

of NO (Nagao and Vanhoutte, 1992). The results of our study show that the mechanical

removal of the endothelium or incubation with the eNOS inhibitor L-NAME significantly

reduced the vasodilator effect of both agonists in Phe contracted vessels, indicating that

these agents require the presence of a functional endothelium and NO to exert their

maximum vasodilator response. Moreover, PPAR-β agonists increased NO production in

HUVECs. Under normal oxidative stress conditions, such as in our experiments, vascular

endothelium-dependent relaxation might be mediated by i) increased endothelial NO

production, ii) potentiation of the NO-cGMP pathway leading to vasorelaxation. This later

mechanism was ruled out because neither L-165041 nor GW0742 modified the relaxant

response induced by the NO donor SNP in endothelium-denuded aorta.

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The NO is formed in the endothelium through the metabolic conversion of L-

arginine to L-citrulline, reaction catalyzed by eNOS. Activation of eNOS is generally a

calcium dependent process (Stuehr, 1997). The raise of intracellular free calcium

concentration, enable calcium-calmodulin binding to eNOS, displacing caveolin-1 and

activating eNOS. However, PPAR-β agonists induced endothelium-dependent

vasorelaxation independent of changes in intracellular calcium concentration because

removal of extracellular calcium and incubation with the intracellular calcium quelator

BAPTA did not alter this relaxant effect. Calcium-independent activation of eNOS can

also be induced by insulin, estrogens and shear stress. Such a mechanism of activation of

eNOS might involve PI3K/Akt pathways, which has recently been shown to phosphorylate

eNOS and alter its sensitivity to calcium so that it is active at sub-physiological

concentrations (Hartell et al., 2005). The incubation of the aortic rings with the PI3K

inhibitor LY-294002 strongly reduced the relaxant responses induced by both PPAR-β

agonists. These results suggest that these agents might activate eNOS by a calcium-

independent PI3K-sensitive pathway. Furthermore, in HUVECs we found an increase of

Akt- and eNOS-phosphorylation at 15 minutes, and that this phosphorylation was inhibited

by the PI3K inhibitor LY-294002, which showed that eNOS activation induced by these

agents is mediated by activation of a PI3K/Akt pathway. In addition, NO production in

HUVECs induced by both PPAR-β agonists was sensitive to PI3K inhibition. These data

support previous results in endothelial progenitor cells describing that PPAR-β agonists

caused activation of the PI3K/Akt pathway within few minutes and in a PPAR-β-

dependent manner (Han et al., 2008).

It is important to note that these compounds may affect vascular tone by acting on

both endothelial cells and smooth muscle cells layers, because, at higher concentrations,

these agents inhibited the contractile response induced by Phe or U46619 in endothelium-

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denuded aorta. The blockade of PPAR-β with the antagonist GSK0660 did not alter the

vasorelaxation induced by both L-165041 and GW0742 in aorta without endothelium

precontracted with Phe, which suggests that these relaxant effects are PPAR-β-

independent. High concentrations of these PPAR-β agonists could inhibit the contractile

apparatus or agonist induced signalling in vascular muscle. The further analysis of the

endothelium-independent relaxation of the PPAR-β agonists indicated that this component

was also independent of extracellular calcium because the phasic transient contraction

induced by Phe in Ca2+-free media, which is expected to be due to Ca2+-release and Ca2+-

sensitization, was strongly reduced. In contrast, the KCl-induced contraction, mainly due

to Ca2+-entry via voltage-dependent channels was almost unaffected. Therefore, the

endothelium-independent relaxation observed at high concentrations of these PPAR-β

agonists seems to share some similarities with the antiaggregant effects of these drugs

reported by Ali et al. (2005) which were also associated to an inhibition of Ca2+ release in

the platelets.

In conclusion, our study shows for the first time that the PPAR-β agonists L-

165041 and GW0742 produce fast, concentration-dependent relaxant effects in rat vascular

tissue. These effects seem to be operated partially via PPAR-β receptors through non-

genomic mechanisms. Relaxation is mostly endothelium- and NO-dependent and it is not

related with the classic Ca2+/calmoduline pathway for eNOS activation but with its

phosphorylation via the PI3K/Akt pathway. The residual endothelium-independent

component was also unaffected by removal of extracellular Ca2+, and seems to be unrelated

to PPAR-β activation.

Perspectives

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The main interesting and novel findings of the present study are that PPAR-β

agonists induce acute vasodilatory effects mediated by the PI3K/Akt/eNOS pathway in

endothelial cells. The activation of the PI3K/Akt pathway by PPAR-β activation has been

reported previously in other cells types such as keratinocytes (Di-Poï et al., 2002) and

endothelial progenitor cells (Han et al., 2008). PPAR-β is a key regulator with potential to

therapeutically target multiple aspects of the metabolic syndrome. The metabolic

syndrome, which clusters as the main metabolic abnormalities central obesity, low

concentrations of plasma high-density lipoprotein (HDL) cholesterol, high levels of

triglycerides, hypertension and hyperglycemia, together with insulin resistance, is

associated with an increased risk of both cardiovascular disease and type 2 diabetes.

PPAR-β activation was known to elevate HDL, lower low-density lipoprotein cholesterol

and triglycerides and suppress hepatic glucose output. The present results, describing

vasodilatory effects of PPAR-β agonists, might be involved in their potential

antihypertensive effects and also collaborate to improve both glucose and insulin deliveries

to target tissues, a major limitation on glucose disposal in hypertension, leading to reduced

insulin resistance.

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References

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Footnotes

This work was supported by Grants from Comisión Interministerial de Ciencia y

Tecnología [SAF2007-62731, AGL2007-66108/ALI, SAF2008-03948], from Junta de

Andalucía, Proyecto de Excelencia [P06-CTS-01555] and by the Ministerio de Ciencia e

Innovación, Instituto de Salud Carlos III [Red HERACLES RD06/0009], Spain. MJZ,

AMQ, PG, RL-S and MR are the holder of a studentship from Spanish Ministerio de

Educación y Ciencia. RJ is a recipient of a “Retorno de Doctores” Program contract, from

Junta de Andalucía (Spain).

RJ and MS are equal contributors to this work.

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Legends for figures:

Figure 1. Vasorelaxant effects of PPAR-β agonists. A) Representative traces of relaxant

effect induced by L165041 (0.1μM – 10 μM) or GW0742 (0.1μM – 30 μM), at 15 minutes

intervals, in phenylephrine (Phe) precontracted intact aortic rings. Concentration-response

curves to the PPAR-β agonists, L165041 (B) and GW0742 (C) were performed in Phe pre-

contracted aortic rings with (+E) or without (-E) functional endothelium and in the

presence of NG-nitro-L-arginine methyl ester (L-NAME, 100 μM, for 20 min). Values are

expressed as mean ± SEM (n = 6-12 rings from different rats). *P<0.05, **P<0.01 vs

control +E rings. The insets show the contractile tension induced by Phe before the

addition of PPAR-β agonist in the different experimental conditions.

Figure 2. Effects of the PPAR- γ antagonist, GW9662, and PPAR-β antagonist GSK0660,

on the vasodilator responses induced by PPAR-β/δ agonists. Aortic rings with (A and B) or

without endothelium (C and D) were incubated in the absence or in the presence of

GW9662 (1 μM) or GSK0660 (1 μM) for 1 h before the addition of phenylephrine (Phe)

and a concentration-response curve to PPAR-β agonists L-165041(A) or GW0742 (B) (0.1

- 30 μM) were carried out in a cumulative fashion. Results are means ± SEM of n = 7-9

experiments. The insets show the contractile tension induced by Phe before the addition of

PPAR-β agonist in the different experimental conditions.

Figure 3. Involvement of Ca2+ on endothelium-dependent vasorelaxant effects of PPAR-β

agonists. The relaxant effects induced by L165041 (0.1-10 μM) (A) or GW0742 (0.1-30

μM) (B) were studied in aortic rings with (+E) or without (-E) endothelium incubated in

normal Krebs solution precontracted to U-46619 (U). In some experiments, a

concentration-response curve to L165041 or GW0742 were performed in intact aortic

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segments incubated in Ca2+-free Krebs solution with or without addition of the intracellular

calcium chelator BAPTA for 30 minutes prior U-46619. Values are expressed as mean ±

SEM (n = 8-14 rings from different rats). *P < 0.05, **P < 0.01 vs +E rings. The insets

show the contractile tension induced by U before the addition of PPAR-β agonist in the

different experimental conditions.

Figure 4. Effect of the PI3K inhibitor, LY-294002, on the vasodilator responses induced

by PPAR-β agonists. Aortic rings with functional endothelium were incubated in the

absence or in the presence for 30 min of LY-294002 (1 μM) and then a concentration-

response curve to L165041 (A) or GW0742 (B) were performed in endothelium-aortic

rings previously contracted with phenylephrine (Phe). Values are expressed as mean ±

SEM (n = 12-15 rings from different rats). *P < 0.05, **P < 0.01vs. control rings. The

insets show the contractile tension induced by Phe before the addition of PPAR-β agonist

in the different experimental conditions.

Figure 5. DAF-2-detected NO released from HUVEC after treatment with A23187 (1 μM)

(A) or L165041 (L, 1, 10 and 30 μM) (B) or GW0742 (GW, 1, 10 and 30 μM) (C). Cells

were washed with phosphate buffered saline (PBS) and then were pre-incubated with L-

arginine (100 μM in PBS, 5 min, 37 ºC). In some experiments, L-NAME (100 μM) was

added 15 min before the addition of L-arginine. The fluorescence was measured at 5, 15

and 30 min. The auto-fluorescence obtained from PBS/DAF-2 was subtracted from each

value. The difference between fluorescence signal without and with L-NAME was

considered NO production. All data are mean ± SEM (n=8). *P < 0.05, **P < 0.01vs time

0. Effects of the HUVECs incubation for 30 min with the PI3K inhibitor LY-294002

(1 μM), or the PPAR-β antagonist GSK0660 (1 μM), or the PPAR-γ antagonist GW9662

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(1 μM) in NO production stimulated by L (30 μM) (D) or by GW (30 μM) (E) measured at

30 min. All data are mean ± SEM (n=8). *P < 0.05, **P < 0.01vs L or GW column.

Figure 6. Effects of L165041 (10 μM) or GW0742 (10 μM) alone or preincubated either

with the PI3K inhibitor LY-294002 (1 μM), or the PPAR-β antagonist GSK0660 (1 μM),

or the PPAR- γ antagonist GW9662 (1 μM) at the protein level of phospho-Akt (A and B)

and phospho- eNOS (C and D) in HUVECs. Results are means ± SEM (n = 4–6) of

densitometric values normalized to the corresponding Akt or to the corresponding eNOS.

**P < 0.01vs control.