Original Contribution Internalization and induction of antioxidant messages by microvesicles contribute to the antiapoptotic effects on human endothelial cells Raffaella Soleti a,b , Emilie Lauret a,b , Ramaroson Andriantsitohaina a,b , Maria Carmen Martı ´nez a,b,n a LUNAM Universite´, Angers, France b INSERM U1063, Stress oxydant et pathologies me´taboliques, Angers, France article info Article history: Received 20 January 2012 Received in revised form 31 August 2012 Accepted 14 September 2012 Available online 23 September 2012 Keywords: Apoptosis Cytoprotection Endothelial cells Microvesicles Reactive oxygen species abstract Microvesicles are plasma membrane-derived fragments released from various cell types during activation and/or apoptosis and posses the ability to deliver biological information between cells. Microvesicles generated from T lymphocytes undergoing activation and apoptosis bear the morphogen Sonic Hedgehog, and exert a beneficial potential effect on the cardiovascular system through their dual capacity to increase nitric oxide and reduce reactive oxygen species production. This study investigated the effect of microvesicles on the apoptosis of human umbilical vein endothelial cells triggered by actinomycin D. Microvesicles prevented apoptosis induced by actinomycin D by modulating reactive oxygen species production: during the early phase of apoptosis, microvesicles might act directly as reactive oxygen species scavengers, owing to their ability to carry active antioxidant enzymes, catalase, and isoforms of the superoxide dismutase. Furthermore, their effects were associated with the ability to increase the expression of manganese-superoxide dismutase in endothelial cells, through the inter- nalization process. Interestingly, microvesicles bearing Sonic Hedgehog induced cytoprotection in endothelial cells through the activation of the Sonic Hedgehog pathway. These findings provide additional evidence that microvesicles from T lymphocytes exert their vasculoprotective effects by promoting internalization and induction of antioxidant messages to the endothelial monolayer. & 2012 Elsevier Inc. All rights reserved. Introduction Endothelial cell (EC) apoptosis represents an essential mechanism acting during development and adulthood; it may contribute to blood-vessel regression, remodeling, morphogenesis, and homeostasis. However, alterations in the regulation of endothelial apoptosis lead to loss of endothelial integrity which is linked to various cardiovascular pathologies such as atherosclerosis, thrombosis, and hind-limb ischemia. Moreover, excessive genera- tion of reactive oxygen species (ROS) coupled with their ability to cause oxidative damage in the vascular endothelium can trigger EC apoptosis [1–4]; endothelial-derived nitric oxide (NO ˙ ) inhibits this process. Thus, EC death can critically disturb the integrity of the endothelial monolayer and may contribute to the initial endothelial injury. Microvesicles (MVs) constitute nonclassical protein secretion pathways by which cells intercommunicate [5]. Their function is strictly linked to the cargo they carry which is, in turn, dependent on the cell type from which they stem and on the stimulation used for their generation. MVs, small particles (0.1–1 mm) released from plasma membrane blebs on activation and/or apoptosis, are able to take part in different physiological and pathophysiological processes [6,7]. Regarding the effects of MVs on apoptosis, few studies are available. It has been demonstrated Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.09.021 Abbreviations: Act D, actinomycin D; ATP, adenosine triphosphate; BSA, bovine serum albumin; CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrroli- dine; Cu/Zn-SOD, copper/zinc superoxide dismutase; CUR61414, N-((3S,5S)-1- (benzo[d][1,3]dioxol-5-ylmethyl)-5-(piperazine-1-carbonyl)pyrrolidin-3-yl)–N- (3-methoxybenzyl)-3,3-dimethylbutanamide; Cycl, cyclopamine; DETC, diethyl- dithiocarbamate; EC(s), endothelial cell(s); EC-SOD, extracellular superoxide dismutase; EGF, epidermal growth factor; EPR, electronic paramagnetic reso- nance; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; FGF-2, fibroblast growth factor 2; H 2 O 2 , hydrogen peroxide; HRP, horseradish peroxidase; HUVECs, human umbilical vein endothelial cells; IGF-1, insulin growth factor 1; L-NA, No-nitro-L-arginine; MAPK, mitogen-activated protein kinase; Mn-SOD, manganese superoxide dismutase; MnTMPyP, manganese(III)tetrakis-(1-methyl- 4-pyridyl)-porphyrin pentachloride; MV(s), microvesicle(s); mRNA, messenger RNA; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase; NO ˙ , nitric oxide; NOS, nitric oxide synthase; PBS, phosphate-buffered saline; PHA, phyto- hemagglutinin; PI, propidium iodide; PI3-k, phosphatidylinositol 3-kinase; PMA, phorbol-12-myristate-13-acetate; ROS, reactive oxygen species; Shh, Sonic Hedgehog; SOD(s), superoxide dismutase(s); TBS, Tris-buffered saline; TBS-T, Tris-buffered saline containing 1% Tween 20; TCTP, translationally controlled tumor protein; TUNEL, terminal deoxynucleotidyltransferase dUTP nick end labeling; VEGF, vascular endothelium growth factor; WST-1, 2-(4-iodophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt n Corresponding author at: INSERM, U1063, Stress oxydant et pathologies me ´ taboliques, Universite ´ d’Angers, Rue des Capucins, Angers, F-49100, France. Fax: þ33 2 44 68 85 88. E-mail address: carmen.martinez@univ-angers.fr (M. Carmen Martı ´nez). Free Radical Biology and Medicine 53 (2012) 2159–2170