Microparticles from Patients with Metabolic Syndrome Induce Vascular Hypo-Reactivity via Fas/Fas-Ligand Pathway in Mice Abdelali Agouni 1,2¤ , Pierre-Henri Ducluzeau 1,3 , Tarek Benameur 1 , Se ´ bastien Faure 1 , Martina Sladkova 1,4 , Lucie Duluc 1 , Georges Leftheriotis 5 , Olga Pechanova 5 , Mirela Delibegovic 2 , Maria Carmen Martinez 1 , Ramaroson Andriantsitohaina 1 * 1 INSERM, U694, Angers, France; Universite ´ d’Angers, Angers, France, 2 Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, Scotland, United Kingdom, 3 De ´partement d’Endocrinologie et Diabe ´tologie, CHU d’Angers, Angers, France, 4 Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic, 5 INSERM, U771, Angers, France Abstract Microparticles are membrane vesicles with pro-inflammatory properties. Circulating levels of microparticles have previously been found to be elevated in patients with metabolic syndrome (MetS). The present study aimed to evaluate the effects of in vivo treatment with microparticles, from patients with MetS and from healthy subjects (HS), on ex vivo vascular function in mice. Microparticles isolated from MetS patients or HS, or a vehicle were intravenously injected into mice, following which vascular reactivity in response to vasoconstrictor agonists was assessed by myography with respect to cyclo-oxygenase pathway, oxidative and nitrosative stress. Injection of microparticles from MetS patients into mice induced vascular hypo- reactivity in response to serotonin. Hypo-reactivity was associated with up-regulation of inducible NO-synthase and increased production of NO, and was reversed by the NO-synthase inhibitor (N G -nitro-L-arginine). The selective COX-2 inhibitor (NS398) reduced the contractile effect of serotonin in aortas from mice treated with vehicle or HS microparticles; however, this was not observed within mice treated with MetS microparticles, probably due to the ability of MetS microparticles to enhance prostacyclin. MetS microparticle-mediated vascular dysfunction was associated with increased reactive oxygen species (ROS) and enhanced expression of the NADPH oxidase subunits. Neutralization of the pro- inflammatory pathway Fas/FasL completely prevented vascular hypo-reactivity and the ability of MetS microparticles to enhance both inducible NO-synthase and monocyte chemoattractant protein-1 (MCP-1). Our data provide evidence that microparticles from MetS patients induce ex vivo vascular dysfunction by increasing both ROS and NO release and by altering cyclo-oxygenase metabolites and MCP-1 through the Fas/FasL pathway. Citation: Agouni A, Ducluzeau P-H, Benameur T, Faure S, Sladkova M, et al. (2011) Microparticles from Patients with Metabolic Syndrome Induce Vascular Hypo- Reactivity via Fas/Fas-Ligand Pathway in Mice. PLoS ONE 6(11): e27809. doi:10.1371/journal.pone.0027809 Editor: Pieter H. Reitsma, Leiden University Medical Center, The Netherlands Received July 6, 2011; Accepted October 25, 2011; Published November 15, 2011 Copyright: ß 2011 Agouni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from Fonds Europe ´en pour le De ´veloppement Re ´gional (R.A. nu 8891), Fondation pour la Recherche Me ´ dicale (R.A. nu INE20050303433 and MCM nu INE20060306500), CNRS, INSERM and Universite ´ d’Angers. A.A. and T.B. are recipients of doctoral fellowships from the French Education Ministry (MENRT). R.A. is supported by a ‘‘Contrat d’Interface’’ INSERM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ramaroson.andriantsitohaina@univ-angers.fr ¤ Current address: Faculty of Health and Medical Sciences, University of Surrey, Guildford, England, United Kingdom Introduction Metabolic syndrome (MetS) is associated with glucose intoler- ance, obesity, ageing, elevated blood pressure and dyslipidaemia, all of which are risk factors associated with cardiovascular morbidity and mortality [1,2]. The prevalence of MetS is increasing and continues to provide challenges for medical research beyond its clinical and public health importance. The pathophysiology of MetS seems to be largely attributable to insulin resistance with the implication of excessive flux of fatty acids [1,2], but also to a pro-inflammatory state resulting from the production of cytokines from adipocytes and macrophages [1,3,4,5]. Thus, increased inflammatory factors and reactive oxygen species (ROS) are associated with detrimental cardiovas- cular alterations linked to MetS. Inflammation is orchestrated by the interactions between inflammatory cells (such as leukocytes) and vascular cells (endothelial and smooth muscle cells) which under activation or apoptosis (for example) lead to the release of circulating microparticles (MPs) [6,7]. MPs are membrane vesicles with pro-coagulant and pro- inflammatory properties [6,7]. The mechanism of MP formation is complex and has yet to be clearly elucidated, due to cell type and stimuli specificity. However, evidence suggest that following cell activation or apoptosis, MP formation occurs due to the sustained elevation in cytosolic calcium concentration in addition to the consequent activation of calpain and protein kinases and the inhibition of phosphatases. In addition to MP formation, these changes result in cytoskeletal reorganization and membrane blebbing [6,8,9,10]. The mechanism of MPs clearance from the circulation is currently unknown, but due to their small size, MPs are believed to be more readily diffusible than cells, and are able to escape phagocytosis [11]. PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27809