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CARDIOVASCULAR

Rosiglitazone, a Peroxisome Proliferator-Activated Receptor- Agonist, Prevents Microparticle-Induced Vascular Hyporeactivity through the Regulation of Proinflammatory Proteins

Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 771, Angers, France (A.T., M.C.M., R.A.); Centre National de la Recherche Scientifique Unité Mixte de Recherche 6214, Angers, France (A.T., M.C.M., R.A.); Université d'Angers, Unité de Formation et de Recherche de Médecine, Angers, France (A.T., M.C.M., R.A.); and Institut Gilbert-Laustriat, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7175-LC1, Faculté de Pharmacie, Illkirch, France (G.A.-M., R.W., S.R.)

Received August 16, 2007; accepted November 21, 2007.

	Abstract

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Microparticles are plasma membrane vesicles with procoagulant and proinflammatory properties. We recently demonstrated that microparticles induce vascular hyporeactivity and evoke up-regulation of proinflammatory protein expression. This study dissected the effect of either in vitro treatment or short-term oral administration of the peroxisome proliferator-activated receptor- (PPAR) agonist, rosiglitazone, on microparticle-induced vascular hyporeactivity of mouse vessels. Microparticles were produced from T cells by actinomycin D treatment. The effects of rosiglitazone on mouse aortic rings incubated with microparticles were investigated. Aortae treated in vitro with rosiglitazone or aortae taken from mice treated by oral administration of the same agonist completely prevented microparticle-induced vascular hyporeactivity in response to U46619 [GenBank] [9,11-dideoxy-11, 9-epoxymethanoprostaglandin F₂). These effects of rosiglitazone occurred independently of the presence of endothelium without modifications in blood parameters. The mechanisms involved abrogation of nitric oxide (NO) and prostacyclin overproduction linked to up-regulation of inducible NO-synthase and cyclooxygenase 2 elicited by microparticles. In addition, rosiglitazone treatment reduced the ability of microparticles to evoke increases in interleukin (IL)-6, IL-8, and nuclear factor (NF)-B transcription, and NF-B expression and activation. These results suggest that rosiglitazone, via PPAR activation, counteracts vascular dysfunction associated with increased release of proinflammatory proteins elicited by microparticles. They underscore therapeutic perspective for rosiglitazone in vascular diseases involving enhanced participation of microparticles.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional factors of a large superfamily of nuclear hormone receptors involved in several biological processes, such as energy homeostasis, cell proliferation and differentiation, fatty acid catabolism, and adipogenesis (Escher and Wahli, 2000). Among the different human PPAR isotypes (Semple et al., 2006), PPAR participates in the transcription of various genes involved in the regulation of lipid metabolism and glucose homeostasis. Moreover, PPAR affects the inflammatory process by preventing foam cell formation-regulating gene expression of transporters implicated in the coordination of cholesterol efflux and the scavenger receptor CD36 of macrophages (Avallone et al., 2006). As a result, PPAR activation inhibits NO overproduction, interleukin (IL)-6, and tumor necrosis factor (TNF)- expression and suppresses COX-2 and iNOS induction by repression of NF-B and activator protein-1 activation (Inoue et al., 2000; Maggi et al., 2000). PPAR can be activated by synthetic agonists belonging the family of thiazolidinediones, for instance rosiglitazone (RZ) (Mohanty et al., 2004). Recent studies performed in diabetic patients have shown that RZ reduces the inflammatory markers (Marfella et al., 2006), and it reduces blood pressure in hypertensive transgenic mice characterized by human renin and angiotensin overexpression (Ryan et al., 2004). These data suggest a protective role for PPAR agonists in cardiovascular diseases because of their anti-inflammatory properties.

The aim of this study was to investigate the potential protective role of PPAR activation on the vascular inflammation. Indeed, the activation of PPAR is known to have anti-inflammatory properties, and thus, the hypothesis that RZ could be used as an anti-inflammatory agent during the vascular inflammation induced by MPs from human T cells has been tested. For this purpose, the effect of RZ was studied after in vitro treatment with the agonist on isolated mouse vessels and in human umbilical arterial smooth muscle cells. Studies were also performed on vessels taken from mice treated by oral administration of the agonist to better mimic the in vivo situation and unmask a preventive role of PPAR activation on inflammatory stimuli such as MPs. In this study, we demonstrate the ability of RZ to protect vessels during or, in a preventive way, before MP treatment.

	Materials and Methods

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MP Production. The human lymphoid CEM T-cell line (American Type Culture Collection number CCL-119; American Type Culture Collection, Manassas, VA) was cultured in free-serum X-vivo15 medium (Cambrex, Verviers, Belgium). Cells (200 x 10⁶) were treated with actinomycin D (0.5 µg/ml, 18 h, 37°C; Sigma-Aldrich, Saint-Quentin Fallavier, France) to induce apoptosis, as described previously (Tesse et al., 2005). The cell supernatant was obtained after two centrifugation steps (750g for 15 min and 200g for 5 min). The supernatant was centrifuged for 45 min at 14,000g. The pelleted MPs were suspended in Hanks' buffer and centrifuged for 45 min at 14,000g. The pellet was recovered in 1 ml of RPMI 1640/M199 medium. MPs isolated from at least five independent preparations were analyzed for each experimental condition. Levels of endotoxin were assessed in all MP preparations with the Limulus amebocyte lysate kit QCL1000 (Cambrex) and were found to be below the lower detection limit of the kit (<0.1 endotoxin U/ml).

MP amounts and their phenotypic characterization were determined by measurement of their procoagulant phosphatidylserine content in a prothrombinase assay described elsewhere, and they were expressed as nanomolar phosphatidylserine equivalent (PS eq) by reference to a calibration curve (Aupeix et al., 1997; Hugel et al., 1999). The MP concentration used in this work is frequently observed in patients with diabetes (Sabatier et al., 2002) and other several pathologies like paroxysmal nocturnal hemoglobinuria (Hugel et al., 1999) or unstable angina (Mallat et al., 2000).

Animals. Animal experiments were performed in accordance with guidelines of the European Community and the French government concerning the use of animals. This study was performed in male Swiss mice (8–10 weeks old). Mice were housed at 20°C with a 12-h light cycle and allowed free access to food and water.

Measurement of Vascular Reactivity. Mice were anesthetized with ketamine (100 mg/kg i.p.; Sigma-Aldrich) plus medetomidine (50 µg/kg i.p.; Pfizer Santé Animale, Paris, France) and killed by decapitation. Aortae were removed, and the adhering fat and connective tissue were carefully cleaned as described previously (Ralay Ranaivo et al., 2004; Ohlmann et al., 2005). Aortic rings of approximately 2 mm, after incubation in a medium with or without MPs, were mounted on a wire myograph filled with physiological salt solution of the following composition: 119 mM NaCl, 4.7 mM KCl, 14.9 mM NaHCO₃, 1.2 mM MgSO₄ · 7H₂O, 2.5 mM CaCl₂, 1.18 mM KH₂PO₄, and 5.5 mM glucose at 37°C and gassed with 95% O₂ and 5% CO₂. Mechanical activity was recorded isometrically with a force transducer (Danish Myo Technology, Aarhus, Denmark). The functionality of the endothelium was tested by the ability of acetylcholine to induce relaxation.

In our previous studies, treatment of vessels in vitro with 30 nM PS eq MPs for 24 h corresponded to the concentration and time required to obtain maximal vascular dysfunction (Martin et al., 2004; Tesse et al., 2005). Thus, all of the experiments in the present work were performed under these conditions.

Vessels with and without functional endothelium were treated in vitro with RZ (5 µM, Avandia; GlaxoSmithKline, Marly-le-Roi, France) for 24 h in the presence or absence of MPs (30 nM PS eq). Supernatants corresponding to the last MPs washing medium have been used as control, and it did not display modifications of vascular reactivity. RZ was used at a maximally active concentration without eliciting cell toxicity (Sauma et al., 2006; Khanolkar et al., 2007). Another group of aortic rings was incubated for 30 min with the selective inhibitor of PPAR, GW9662 (1 µM; Sigma-Aldrich), before treatment with MPs plus RZ for 24 h. Afterward, contraction experiments were conducted. GW9662 was used at a concentration that inhibits the CD36 induction by IL-4-dependent PPAR activation in human peritoneal macrophages as previously reported by Huang et al. (1999).

In another set of experiments, mice were treated by gavage either with 30 mg/kg/day of RZ for a period of 1 week or an equal volume of physiological solution (control mice). At this dose, in vivo treatment with RZ did not significantly modify glycemia and metabolic blood parameters in mice (Table 1). After treatment with either RZ or vehicle, mice were sacrificed, and aorta was harvested. Aortic rings without endothelium were then incubated in vitro for 24 h in a medium with or without 30 nM PS eq of MPs.

Concentration-response curves were constructed by cumulative application of U46619 [GenBank] (1 nM–1 µM, Calbiochem, Nottingham, UK) to vessels with or without functional endothelium and, after 30 min preincubation, with either the NO-synthase inhibitor, N^G-nitro-L-arginine (L-NA; Sigma-Aldrich), the selective COX-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398; Sigma-Aldrich), or L-NA plus NS-398.

NO Spin Trapping and Electronic Paramagnetic Resonance Studies. Detection of NO production was performed using a previously described technique with Fe²⁺ diethyldithiocarbamate (DETC; Sigma-Aldrich) as a spin trap (Mülsch et al., 1995). The U46619 [GenBank] -treated vessels were placed in 24-well clusters filled with 250 µl of physiological salt solution and incubated with 250 µlof colloid Fe(DETC)₂ (37°C, 1 h). These studies were performed on a table-top X-band spectrometer MiniScope (Magnettech, Berlin, Germany). Recordings were made at 77°K, using a Dewar flask. Instrument settings were as follows: 10 mW of microwave power, 1 mT of amplitude modulation, 100 kHz of modulation frequency, 150 s of sweep time, and 3 scans.

Determination of Prostanoid Production. Intact vessels were treated with U46619 [GenBank] (1 µM, 20 min at 37°C). The medium was next collected. Then, 6-keto-prostaglandin (PG)F₁ was measured by an enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI). The concentration of 6-keto-PGF₁ was expressed as picomoles per milligram of tissue (dry weight).

Staining and Imaging by Confocal Microscopy. Vessels with endothelium were frozen and cut into 10-µm sections. Fixed sections were incubated (2 h) in blocking buffer (5% nonfat dry milk in phosphate-buffed saline). After three washes, tissue sections were incubated overnight with monoclonal murine anti-iNOS (1:50; Transduction Laboratories, Heidelberg, Germany) or anti-COX-2 (1: 100; Transduction Laboratories) antibodies. For NF-B RelA/p65 immunostaining, we used a polyclonal NF-B p65 antibody (1:100; Abcam, Cambridge, UK). Three washes were followed by incubation (1 h) with murine Alexa Fluor-488-labeled antibody (1:100; Molecular Probes, Leiden, The Netherlands) for iNOS and COX-2 immunostaining or with rabbit Alexa Fluor-488-labeled antibody (1:100; Molecular Probes) for NF-B p65 immunostaining, respectively. After the washes, the vessel sections were mounted onto glass slides. The MRC-1024ES confocal equipment mounted on a Nikon Eclipse TE 300 inverted microscope was used for the optical sectioning of the tissue. Digital image recording was performed using LaserSharp Software (Bio-Rad, Hercules, CA). Confocal Assistant was used for image analysis (TC Brelje, Minneapolis, MN).

Western Blot Analysis. After 24 h of incubation in a medium with MPs alone, MPs plus RZ, or without MPs, crushed aortae were homogenized and lysed. Seventy-five micrograms of proteins were separated on 8% SDS-polyacrylamide gel electrophoresis. Blots were probed with phosphorylated IB- (US Biological, Swampscott, MA) or NF-B p65 antibodies followed by the anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody, respectively. Protein levels were normalized using a mouse monoclonal antibody against -tubulin.

Human Smooth Muscle Cell Culture. Human umbilical artery smooth muscle cells from PromoCell GmbH (Heidelberg, Germany) were cultured with MCDB 131 (Invitrogen, Cergy Pontoise, France) (Knedler and Ham, 1987) supplemented with 10% bovine serum (Invitrogen, Cergy-Pontoise, France), 2 mM L-glutamine, 100 U/ml penicillin, and 100 ng/ml streptomycin. Cells showed a constant phenotype during subculturing and were used on passage 3.

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis. Human umbilical artery smooth muscle cells were stimulated for 24 h with or without MPs or MPs plus RZ. Cells were detached using trypsin, and after two subsequent steps of centrifugation at 500g for 10 min, the pellet containing cells were frozen in liquid N₂ and used to investigate the expression of mRNA for IL-6, IL-8, IL-1β, TNF-, and for the transcription factor NF-B p65 by real-time reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR analyses were carried out by Service Commun de Cytométrie et d'Analyses Nucléotidiques from Angers University, using a Chromo 4 (Bio-Rad, Marnes-la-Coquete, France) and SYBR Green detection. Primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Quantifications were realized according to the C_t method, and the relative gene-expression levels were normalized using the geometric mean of three housekeeping genes as described previously (Vandesompele et al., 2002).

Data Analysis. Data are represented as the mean ± S.E.M., and n represents the number of animals. EC₅₀ and 95% confidence intervals were expressed in nanomolars, and they were calculated using GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego, CA). Statistical analysis was performed by a one-way analysis of variance, Kruskal-Wallis and Mann-Whitney U tests, or two-way analysis of variance for repeated measurements, and a subsequent Tukey post-hoc test was performed with the StatView 5.0 software (SAS Institute, Cary, NC). P < 0.05 was considered to be statistically significant.

	Results

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In Vitro Incubation with RZ Restores the MP-Induced Vascular Hyporeactivity to U46619 [GenBank] via PPAR Activation. U46619 [GenBank] produced a concentration-dependent increase in the tension of aortic rings in vessels with and without functional endothelium (Fig. 1, A–C). As described previously (Tesse et al., 2005), incubation of mouse aortic rings with 30 nM PS eq MPs for 24 h decreased vascular reactivity to the agonist in vessels with and without functional endothelium (Fig. 1, A and B). In vitro incubation with RZ reversed vascular hyporeactivity induced by MPs without affecting contraction to U46619 [GenBank] in control vessels (Fig. 1, A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Effect of in vitro treatment of mouse aortae with RZ (5 µM) on MP-induced vascular changes. The image shows concentration-response curves to U46619 of mouse aortic rings with functional endothelium (A) or without endothelium (B) after 24-h incubation with either RPMI 1640/M199 medium (controls, n = 5, ), medium containing either 30 nM PS eq of MPs (n = 5, ), RZ (5 µM, n = 5, ), or MPs plus RZ (MPs+RZ, n = 5, ). C, The graphic shows concentration-response curves to U46619 of mouse aortic rings without functional endothelium after 24-h incubation with either RPMI 1640/M199 medium (controls, n = 5, ), 30 nM PS eq of MPs (n = 5, ), or 24-h incubation with either GW9662 (1 µM, n = 5, ) or 30 nM PS eq of MPs plus GW9662 (MPs+GW9662, 1 µM, n = 5, ). EC50 and 95% confidence intervals (in brackets) were expressed in nanomolars. EC50 values were not significantly modified by any treatment. D, quantification of NO production by the NO-Fe(DETC) signal with EPR in units/weight in milligrams of dried sample (A/Wds) (n = 5). E, production of 6-keto PGF1 in mice aortae exposed to U46619 (1 µM). The quantification of NO or 6-keto PGF1 was obtained in mouse aortae after incubation with either RPMI 1640/M199 medium (control, n = 5), 30 nM PS eq of MPs (n = 5), RZ (5 µM, n = 5), or MPs plus RZ (MPs+RZ, n = 5). *, P < 0.05 significantly different from control; , P < 0.05 and , P < 0.01 significantly different from MPs. The 6-keto PGF1 production was not significantly different between the control and RZ-treated vessels.

To investigate the effect of RZ on NO and COX metabolite productions that are involved in vascular hyporeactivity induced by MPs (Tesse et al., 2005), direct measurements of NO and prostacyclin were performed in control and MP-treated aortae in the absence or presence of RZ. In situ measurements of NO production were performed by electronic paramagnetic resonance (EPR) spectroscopy using Fe-(DETC)₂ as a spin trap. The NO-Fe(DETC)₂ EPR signal was greater in aortae treated with MPs compared with nontreated vessels (Fig. 1D). It is interesting to note that in vitro treatment with RZ alone did not modify NO production, but it reduced NO overproduction evoked by MP treatment toward that of control (Fig. 1D).

Measurement of aortic production of 6-keto-PGF₁, the stable product of PGI₂ showed that MP treatment increased 6-keto-PGF₁ compared with control aortae (Fig. 1E), confirming results obtained in our previous work (Tesse et al., 2005). In this study, we found that RZ alone did not significantly modify 6-keto-PGF₁ in control vessels but prevented its overproduction after MP treatment (Fig. 1E).

In Vitro Incubation with RZ Represses Expression and Activation of NF-B Induced by MPs. Because enhanced expression of proinflammatory enzymes and subsequent NO and prostaglandin overproduction is under the control of the NF-B/Rel family of transcription factors, immunohistochemical studies for the p65/RelA subunit, which only has transactivation domains capable of initiating transcription, were performed. Weak or no specific staining was found in control aortae (Fig. 2A). Marked aortic staining of the p65/RelA subunit of NF-B was found in aortae incubated with MPs (Fig. 2B). In vitro treatment with RZ strongly reduced p65/RelA subunit staining in aortae incubated with MPs (Fig. 2C). Negative controls obtained by incubation with the secondary rabbit fluorescence-labeled antibody did not display any staining (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Immunohistochemical staining for NF-B p65 of mouse aortae after incubation with either RPMI 1640/M199 medium (A; n = 3), 30 nM PS eq of MPs (B; n = 3) or MPs plus RZ (C; n = 3). D, The image shows Western blotting for NF-B p65 and phosphorylated IB- expression in mouse aortae after 24-h incubation with either RPMI 1640/M199 medium (controls, n = 4), 30 nM PS eq of MPs (n = 4), or MPs plus RZ (MPs+RZ, n = 4). Scale bar, 150 µm. E, The image shows quantitative real-time RT-PCR analysis of the normalized relative quantity of NF-B p65 mRNA expression in human umbilical artery smooth muscle cells after 24-h incubation with either RPMI 1640/M199 medium (controls, n = 4), 30 nM PS eq of MPs (n = 6), or MPs plus RZ (MPs+RZ, n = 4). **, P < 0.01 significantly different from control; , P < 0.05 significantly different from MP-treated cells.

RZ Prevents the Increase of IL-6 and IL-8 mRNA Expression Induced by MPs. To determine whether MP treatment regulates other inflammatory genes and the effect of RZ in MP-induced transcriptional regulation, determinations by RT-PCR of IL-6, IL-8, IL-1β, and TNF- mRNA expression were performed in human smooth muscle cells. No expression of TNF- mRNA in control, MP-treated, or MPs plus RZ-treated cells could be detected under the experimental conditions used (data not shown). With regard to IL-6, IL-8, and IL-1β, MPs were able to significantly increase IL-6 and IL-8 mRNA expression, and these effects were completely reversed by RZ (Fig. 3, A and B). IL-1β mRNA expression was not significantly modified by MPs alone or MPs plus RZ (Fig. 3C).

View larger version (9K): [in this window] [in a new window]   Fig. 3. Quantitative real-time RT-PCR analysis of normalized relative quantity of IL-6 (A), IL-8 (B), and IL-1β (C) mRNA expressions in human umbilical artery smooth muscle cells after 24-h incubation with either RPMI 1640/M199 medium (controls, n = 4), 30 nM PS eq of MPs (n = 6), or MPs plus RZ (MPs+RZ, n = 4). *, P < 0.05 significantly different from control; , P < 0.05 significantly different from MP-treated cells.

The Oral Treatment with RZ Prevents MP-Induced Vascular Hyporeactivity. Because the effects of MPs occurred directly in smooth muscle cells, experiments were carried out in endothelium-denuded preparations. The incubation of mice aortic rings with 30 nM PS eq MPs for 24 h decreased vascular reactivity to U46619 [GenBank] in the endothelium-denuded vessels of mice treated with the physiological solution (Fig. 4A). Oral administration of mice with RZ did not significantly alter the contraction to U46619 [GenBank] , but it prevented the vascular hyporeactivity evoked by MPs (Fig. 4B).

View larger version (10K): [in this window] [in a new window]   Fig. 4. Effect of in vivo treatment with RZ. The image shows concentration-response curves to U46619 of aortic rings without functional endothelium, from mice treated for 1 week of gavage with physiological solution (A) or with RZ (B), and then incubated for 24 h with RPMI 1640/M199 medium (control, n = 4, ), 30 nM PS eq of MPs (n = 4, ), RZ (5 µM, n = 5, ), or MPs plus RZ (MPs+RZ, n = 5, ). **, P < 0.01 significantly different from control. EC50 and 95% confidence intervals (in brackets) were expressed in nanomolars. EC50 values were not significantly modified by any treatment.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effect of L-NA and NS-398 on vascular reactivity after in vivo treatment of RZ. A and B, The graphic illustrates the effect of L-NA on U46619-induced vasoconstriction in aortic rings incubated with 30 nM PS eq of MPs from mice treated with either physiological solution (A; n = 4) or RZ (B; n = 4). C and D, The image shows the effect of NS-398 or L-NA plus NS-398 on U46619-induced vasoconstriction in aortic rings incubated with MPs from mice treated with either physiological solution (C; n = 4) or RZ (D; n = 4). E and F, The image shows the effect of L-NA or NS-398 or L-NA plus NS-398 on U46619-induced vasoconstriction in aortic rings from mice treated in vivo with physiological solution or 30 mg/kg/day of RZ. *, P < 0.05 and **, P < 0.01 significantly different from MP-treated vessels. EC50 and 95% confidence intervals (in brackets) were expressed in nanomolars. EC50 values were not significantly modified by any treatment.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effect of MPs and RZ on iNOS and COX-2 expression and on NF-B p65/RelA activation. The image shows immunohistochemical staining for iNOS, COX-2, and NF-B p65 in aortic rings after incubation with either RPMI 1640/M199 medium (A, D, G; control, n = 3) or 30 nM PS eq of MPs in vessels from mice treated in vivo either with physiological solution (B, E, H; n = 3) or RZ (C, F, I; n = 3). Scale bar, 100 µm.

	Discussion

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We have recently reported that MPs from apoptotic human T cells are able to induce vascular hyporeactivity to vasoconstrictor agonists by inducing the overproduction of NO and prostacyclin. These effects were subsequent to the up-regulation of proinflammatory protein expression (iNOS and COX-2) through NF-B-dependent transcription (Tesse et al., 2005). MP-induced vascular hyporeactivity associated with vascular inflammation is not linked to an increase of endothelial derived NO known to possess an antihypertensive property; it is rather due to a deleterious role of MPs from human T cells on vascular reactivity as a consequence of NO derived from iNOS. In the present study, we confirm these results, and, most interestingly, we provide direct evidence for a direct protective role of PPAR activation by RZ against the proinflammatory mechanisms by which MPs from T cells impair vascular reactivity. Indeed, in vitro or oral administration of RZ completely prevented MP-induced vascular hyporeactivity in response to U46619. [GenBank] The effect of RZ was independent of the presence of functional endothelium on MP-treated vessels, and it was strictly linked to its ability to activate PPAR on vascular cells. Indeed, the selective inhibitor of PPAR, GW9662, completely abolished the protective role of RZ on the hyporeactivity induced by MPs. Moreover, in vitro RZ treatment prevents NO and prostacyclin overproduction by inhibiting MP-induced iNOS and COX-2 expression. In addition, the effect of MPs on NF-B p65/RelA expression and/or activation and phosphorylation of IB- (essential for the release of active NF-B p65/RelA and its translocation in the nucleus) were completely prevented by RZ. Because NF-B p65/RelA mRNA levels and phosphorylation of IB alpha were significantly modified by MP treatment, it is plausible to suggest that MPs regulate the NF-B pathway at both transcriptional and post-transcriptional levels.

The protective effects of RZ were also obtained by oral administration of the drug at a dose at which it slightly, but not significantly, modified glycemic and metabolic blood parameters in mice. These results suggest that the protective property of RZ occurs independently of its ability to regulate lipid metabolism.

Several clinical or animal studies (Minamikawa et al., 1998; Li et al., 2000; Boden and Zhang, 2006; Marfella et al., 2006) reported that the treatment with glitazones reduced the inflammatory responses leading to atherogenesis in blood vessels. In addition, it is known that PPAR agonists can inhibit the expression of proinflammatory cytokines, such as TNF-, IL-1β, IL-6, and IL-12, in lipopolysaccharide-stimulated monocytes/macrophages (Jiang et al., 1998; Chung et al., 2000). Moreover, PPAR activation regulates chemotaxis on the vascular wall and the adhesion of leukocytes to endothelial cells (Murao et al., 1999). Altogether, these data underline the role played by PPAR receptors in the inflammatory process. It is interesting to note that the present study provides evidence that MPs deriving from human leukocytes can induce the up-regulation of IL-6 and IL-8 mRNA expression in agreement with previous works (Mesri and Altieri, 1998), demonstrating that RZ prevented these effects through PPAR activation.

A recent study has shown that pioglitazone, another PPAR ligand, reduces the circulating level of endothelial MPs in the metabolic syndrome (Esposito et al., 2006) and that this effect is independent of its ability to ameliorate insulin sensitivity. These authors hypothesized that the effect of pioglitazone on MP levels might be linked to the anti-inflammatory effect of PPAR ligands. However, the mechanisms implicated in these effects are not yet elucidated. In addition to the potential effect of PPAR ligands to reduce circulating levels of MPs, in the present study we showed that RZ, regardless of the route of administration (in vitro incubation or oral treatment), restores the MP-induced hyporeactivity to U46619 [GenBank] in vessels with or without functional endothelium. Thus, it is likely that RZ mediates its action directly on the vessel wall.

RZ treatment prevents NO and prostacyclin overproduction induced by MPs from human T cells. The present study shows that RZ treatment inhibits iNOS and COX-2 expression in MP-treated vessels as evidenced by immunostaining detected by confocal microscopy. In accordance with our study, it has been reported that PPAR agonists reduce NO production via inhibition of iNOS in lipopolysaccharide- or interferon -stimulated RAW264 macrophages (Colville-Nash et al., 1998). Other authors have shown that RZ decreases iNOS and COX-2 expression in lungs from rats treated with carrageenan, a proinflammatory substance (Cuzzocrea et al., 2004). This protective effect is probably linked to the ability of RZ to inhibit the NF-B activation in the vessel wall. Several mechanisms have been proposed to explain how PPAR ligands can inhibit NF-B pathway. Ligands for PPAR can induce a physical interaction of PPAR with p65, leading to the inhibition of NF-B (Chen et al., 2003). Other authors suggest the SUMOylation of the PPAR ligand-binding domain, which mediates transrepression of inflammatory response genes by PPAR ligands (Pascual et al., 2005). By contrast, Trifilieff et al. (2003) have shown that although PPAR ligands are able to reduce inflammation, this effect might not be mediated by antagonism of the NF-B pathway. In the present study, we demonstrate that MPs from human T cells exert their effects on NF-B cascade at mRNA and protein expressions. In addition, RZ prevented the effect of lymphocytic MPs at both the transcriptional and post-transcriptional levels in smooth muscle cells.

In summary, RZ prevents the ability of MPs from human T cells to evoke IL-6 and IL-8 production and NF-B expression and activation, which in turn up-regulates iNOS and COX-2 expression leading to the production and release of the vasodilatory factors, NO and prostacyclin. These effects result from a direct action of RZ on smooth muscle cells via PPAR activation. Thus, these results show that PPAR agonist may be a potential therapeutic approach to fight against vascular dysfunction evoked by MPs accompanying inflammatory diseases.

	Acknowledgements

 
We thank F. Zobairi for help in MP preparation. We also thank L. Preisser and M.-H. Guilleux from Service Commun de Cytométrie et d'Analyses Nucléotidiques from Institut Fédératif de Recherche 132 (Univesité d'Angers) for assistance in RT-PCR quantification.

	Footnotes

 
G.A.-M. is a recipient of a Fellowship from the Atomic Energy Commission of Syria. This work was supported in part by grants from Fondation pour la Recherche Médicale (Grant INE20050303433; to R.A.), Fonds Européen pour le Développement Régional (Grant 8891; to R.A.), and Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, and Université d'Angers.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.130278.

ABBREVIATIONS: MPs, microparticles; NO, nitric oxide; iNOS, inducible NO-synthase; GW9662, 2-chloro-5-nitro-N-phenylbenzamide; COX-2, cyclooxygenase 2; NF, nuclear factor; PPARs, peroxisome proliferator-activated receptors; IL, interleukin; TNF, tumor necrosis factor; RZ, rosiglitazone; PS eq, phosphatidylserine equivalent; L-NA, N^G-nitro-L-arginine; EPR, electronic paramagnetic resonance; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide; DETC, diethyldithiocarbamate; PG, prostaglandin; RT-PCR, reverse transcription-polymerase chain reaction; U46619 [GenBank] , 9,11-dideoxy-11, 9-epoxymethanoprostaglandin F₂.

Address correspondence to: Dr. Ramaroson Andriantsitohaina, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 771-Centre National de la Recherche Scientifique Unité Mixte de Recherche 6214, Faculté de Médecine, rue Haute de Reculée, 49045 Angers, France. E-mail: ramaroson.andriantsitohaina{at}univ-angers.fr

	References

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