Endothelium-Dependent Vasodilator Effects of Peroxisome Proliferator-Activated Receptor Agonists via the Phosphatidyl-Inositol-3 Kinase-Akt Pathway

Peroxisome proliferator-activated receptor / (PPAR) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily that regulates the transcription of many target genes. More recently, acute, nongenomic effects of PPARagonists have also been described. In the present study, we hypothesized that PPARagonists might exert acute nongenomic effects on vascular tone. Here, we report that the structurally unrelated PPARligands [4-[3-(4-acetyl-3-hydroxy-2propylphenoxy)propoxy]phenoxy]acetic acid (L-165041) and 4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl] methyl]thio]-2-methylphenoxy]acetic acid (GW0742) induced vascular relaxation in phenylephrine-precontracted endotheliumintact rat aortic rings, which was significantly inhibited by endothelial denudation or nitric-oxide synthase (NOS) inhibition with N-nitro-L-arginine methylester. These relaxant effects reached steady state within 15 min. The relaxation induced by L-165041 and GW0742 in aortic rings precontracted with the thromboxane A2 analog 9,11-dideoxy-11 ,9 -epoxymethanoprostaglandin F2 (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)ethaneN,N,N ,N -tetraacetic acid tetra(acetoxymethyl) ester. However, the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) inhibited the endothelium-dependent relaxant responses induced by both PPARagonists. Blockade of PPARwith 3-[[[2-methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylic acid methyl ester (GSK0660) also partially inhibited these relaxant responses, although PPARblockade with 2-chloro-5-nitro-N-phenylbenzamide (GW9662) had no effect. In human umbilical vein endothelial cells, L-165041 and GW0742 increased nitric oxide (NO) production and Akt and endothelial NOS (eNOS) phosphorylation, which were sensitive to PI3K inhibition and PPARblockade. In conclusion, the PPARagonists acutely caused vasodilatation, which was partially dependent on endothelial-derived NO. The eNOS activation is calciumindependent and seems to be related to activation of the PI3KAkt-eNOS pathway. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors that heterodimerize with the retinoid X receptor to regulate the transcription of diverse genes (Kota et al., 2005). There are three known PPAR subtypes: , (also referred to as ), and . PPAR, 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, expressed principally in adipose tissue, liver, and vascular and immune This work was supported in part by Comisión Interministerial de Ciencia y Tecnología [Grants SAF2007-62731, AGL2007-66108/ALI, SAF2008-03948]; Junta de Andalucía, Proyecto de Excelencia [P06-CTS-01555]; and the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III [Red HERACLES RD06/ 0009], Spain. R.J. and M.S. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.159806. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; VSMC, vascular smooth muscle cell; HUVEC, human umbilical vein endothelial cell; L-165041, [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy] phenoxy]acetic acid; GW0742, 4-[[[2-[3-fluoro-4-(trifluoromethyl) phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]acetic acid; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; L-NAME, Nnitro-L-arginine methylester; U-46619, 9,11-dideoxy-11 ,9 -epoxymethanoprostaglandin F2 ; BAPTA, 1,2-bis-(o-aminophenoxy)ethaneN,N,N ,N -tetraacetic acid; PI3K, phosphatidylinositol 3-kinase; LY-294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; GSK0660, 3-[[[2-methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylic acid methyl ester; GW9662, 2-chloro-5-nitro-N-phenylbenzamide; PBS, physiological buffer saline; Akt, protein kinase B; DAF-2, diaminofluorescein-2; A23187, calcium ionophore calcimycin. 0022-3565/10/3322-554–561$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 2 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 159806/3552901 JPET 332:554–561, 2010 Printed in U.S.A. 554 http://jpet.aspetjournals.org/content/suppl/2009/11/11/jpet.109.159806.DC1 Supplemental material to this article can be found at: at A PE T Jornals on O cber 7, 2017 jpet.asjournals.org D ow nladed from cells, is related to adipogenesis and glucose homeostasis and is activated by thiazolidinediones. Although PPARis 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). PPARand/or PPARligands 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 favorable cardiovascular profile as a result of their well-known antiatherosclerotic, anti-inflammatory, and vasodilator effects (Schiffrin et al., 2003), as well as their abilities to inhibit endothelial and vascular smooth muscle cell (VSMC) proliferation (Benkirane et al., 2006; Lee et al., 2006), to reduce cardiac hypertrophy (Asakawa et al., 2002), inhibit platelet aggregation (Moraes et al., 2007), and decrease blood pressure (Iglarz et al., 2003; Khan et al., 2005). PPARligands have been more recently developed and are currently in clinical trials for the treatment of dyslipidemia (Barish et al., 2006; Risérus et al., 2008). However, less is known about other nonmetabolic effects of these drugs. Thus far, it has been shown that PPARactivation improves cardiac 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); however, paradoxically, it induces endothelial cell proliferation and angiogenesis (Piqueras et al., 2007; Han et al., 2008). More recently, multiple nongenomic effects have been reported for agonists of different nuclear receptors (Burgermeister and Seger, 2007; Cheskis et al., 2007; Stahn and Buttgereit, 2008). Thus, PPARligands inhibit the nuclear factor B (Chen et al., 2003), or retinoid X receptor ligands exert antiaggregant effects (Moraes et al., 2007). PPARagonists also inhibit proliferation and migration of rat VSMC (Han et al., 2008) and inhibit platelet aggregation and activation (Ali et al., 2006) via nongenomic effects. We hypothesized that PPARagonists could exert vasodilatory effects via nongenomic mechanisms. Because of the promising therapeutic role of PPARagonists and the relative lack of knowledge of the actions of PPARin the vascular territory, in this study we investigated the short-term effects of L-165041, a weak nonselective PPARagonist (Willson et al., 2000), and GW0742, a selective PPARagonist (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 NO synthase (eNOS) in HUVECs. A preliminary account of these results was presented at the 2007 Winter Meeting of the British Pharmacological Society (Jimenez et al., 2007). Materials and Methods Tissue Preparation and Measurement of Tension. The investigation conforms with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 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 (118 mM NaCl, 4.75 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 2 mM CaCl2, 1.2 mM KH2PO4, and 11 mM glucose) 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 an isometric forcedisplacement transducer (Letigraph 2000; Letica S.A., Barcelona, Spain), respectively, as described previously (Jiménez et al., 2007), and equilibrated for 60 to 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. For the experiments in which Ca -free Krebs’ solution was used, CaCl2 was omitted and 0.5 mM 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 the experimental conditions. When contractions were stable, concentration-relaxant response curves were carried out by cumulative addition of the PPARagonists L-165041 or GW0742 (0.1–30 M) at 15-min intervals. Addition of vehicle (dimethyl sulfoxide, 0.1%) had no significant relaxant effect (2 3% relaxation at the highest concentration of dimethyl sulfoxide tested, n 6). In some endothelium-intact aortic rings, the same protocol was performed in the presence of the eNOS inhibitor N-nitro-L-arginine methylester (LNAME; 10 4 M) for 20 min. To explore whether PPARagonists 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 6 M) in the absence or presence of L-165041 or GW0742 (10 M) in endothelium-denuded aortic rings previously contracted with Phe. To examine the involvement of Ca on the endothelium-dependent relaxation induced by the PPARagonists, two experimental protocols were performed: 1) to test the role of extracellular Ca , intact aortic segments were incubated in Ca -free Krebs’ solution for 30 min before the addition of U-46619 (0.1 M), a Ca -independent vasoconstrictor agent, and then a concentration-response curve to L-165041 or GW0742 (0.1–30 M) was constructed; 2) to test the role of intracellular Ca , the relaxant responses induced by these agents were analyzed in aortic rings incubated for 30 min with the intracellular calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid (BAPTA) tetra (acetoxymethyl) ester (10 5 M) and, after washing for 15 min, precontracted with U-46619 (10 M) in Ca free Krebs’ solution. To evaluate the involvement of phosphatidyl-inositol-3 kinase (PI3K) in the relaxant effects of the PPARagonists, some aortic rings with endothelium were incubated for 30 min in Krebs’ solution containing the PI3K inhibitor LY-294002 (1 M). Then the vessels were exposed to Phe (1 M), and when the contractile response was stable, a concentration-response curve to L-165041 or GW0742 was constructed. To show whether these relaxant effects were related to PPARor PPARactivation, the effects of L-165041 or GW0742 in Phe-precontracted rings in the presence of the PPARantagonist GSK0660 (1 M) or the PPARantagonist GW9662 (1 M) were investigated. Protein Phosphorylation in HUVECs. HUVECs were extracted from umbilical cords (modified from Vargas et al., 1994). In brief, 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 2 mM penicillin/ streptomycin 2 mM amphotericin B 2 mM glutamine 10 mM HEPES 30 g/ml endothelial cell growth supplement 100 mg/ml Vasodilator Effects of PPARAgonists 555 at A PE T Jornals on O cber 7, 2017 jpet.asjournals.org D ow nladed from heparin). The collected cells were seeded in culture flasks pretreated with gelatin 0.2% containing culture medium. To perform the Western blots, cells were washed and incubated 3 h only with medium. Then they were incubated with L-165041 or GW0742 (10 M) for 15 min, and some with LY-294002 (1 M) or PPARantagonist GSK0660 (1 M) or GW9662 (1 M) 30 min before and during the PPARagonist exposure. After this period, cells were washed with PBS and homogenized. Western blots were performed with 30 g of protein/lane, previously determined by the bicinchoninic acid assay (Walker, 1994). SDSpolyacrylamide gel electrophoresis (8%) was performed in a mini-gel system (Bio-Rad Laboratories, Hercules, CA). The proteins were transferred to polyvinylidene difluoride membranes for 1 h and were then blocked with Tris-buffered saline (containing 0.1% Tween 20) containing 5% nonfat dry milk for 90 min at room temperature. Phosphorylated protein kinase B (Akt) (Ser473), phosphorylated eNOS (Ser1177), Akt, and eNOS were detected after the membranes were incubated with the respective primary antibodies (rabbit anti-p-eNOS-Ser1177, mouse anti-eNOS, rabbit anti-p-Akt-Ser473, and rabbit anti-Akt, 1:1000 dilution) overnight at 4°C. The membranes were then washed three times for 10 min in Tris-buffered saline (containing 0.1% Tween 20) and incubated with secondary peroxidase conjugated goat anti-rabbit or goat anti-mouse antibodies (1:2500), respectively. All the incubations were performed at room temperature for 2 h. 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 ratios were calculated, and data are expressed as a percentage of the values in control cells from the same gel. Quantification of NO 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). In brief, 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 preincubated with Larginine (100 M in PBS, 5 min, 37°C). In some experiments, LNAME (10 4 M) was added 15 min before the addition of L-arginine. Subsequently, DAF-2 (0.1 M) and either the calcium ionophore calcimycin [(A23187) 1 M] or the PPARagonist L-165041 (1, 10, and 30 M) or GW0742 (1, 10, and 30 M) were added, and cells were incubated in the dark at 37°C. Then the fluorescence was measured at 5, 15, and 30 min, respectively, using a spectrofluorometer (Fluorostart; BMG Labtechnologies, Offenburg, Germany) with excitation wavelength set at 495 nm and emission wavelength at 515 nm. The autofluorescence obtained from PBS/DAF-2 was subtracted from each value. In some experiments, the fluorescence signal induced by L-165041 or GW0742 (30 M) was measured in HUVECs pretreated for 30 min with LY-294002 (1 M), GSK0660 (1 M), or GW9662 (1 M). Materials. L-165041, GW0742, and GSK0660 were purchased from Tocris Bioscience (Bristol, UK). Medium 199 and HEPES were obtained from Lonza Verviers SPRL (Verviers, Belgium). Rabbit anti-p-eNOS-Ser1177, mouse anti-eNOS, rabbit anti-p-Akt-Ser473, and rabbit anti-Akt were from Cell Signaling Technology Inc. (Danvers, MA). Secondary peroxidase conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Electrochemiluminescent system was from GE Healthcare (Little Chalfont, Buckinghamshire, UK). The rest of the products were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Statistical Analysis. Results are expressed as mean S.E.M., and n reflects the number of experiments performed. Statistically significant differences between groups were calculated by an analysis of variance followed by a Newman-Keuls test. P 0.05 was considered statistically significant.