Laffont PLT MP micro RNAs Blood 2013 122 253

doi:10.1182/blood-2013-03-492801Prepublished online May 7, 2013;2013 122: 253-261

Patrick ProvostBenoit Laffont, Aurélie Corduan, Hélène Plé, Anne-Claire Duchez, Nathalie Cloutier, Eric Boilard and complexes to endothelial cells via microparticles

microRNA•Activated platelets can deliver mRNA regulatory Ago2

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Regular Article

PLATELETS AND THROMBOPOIESIS

Activated platelets can deliver mRNA regulatory Ago2•microRNAcomplexes to endothelial cells via microparticlesBenoit Laffont,1,2 Aurelie Corduan,1,2 Helene Ple,1,2 Anne-Claire Duchez,1,2 Nathalie Cloutier,1,2 Eric Boilard,1,2

and Patrick Provost1,2

1Centre Hospitalier Universitaire de Quebec Research Center/Centre Hospitalier de l’Universite Laval, Quebec, Canada; and 2Faculty of Medicine, Universite

Laval, Quebec, Canada

Key Points

• Activated platelets releasemicroRNA miR-223preferentially through MPsthat can be internalized byendothelial cells.

• Platelet MP-derivedAgo2�microRNA complexesare functional and canregulate endogenous geneexpression in recipientendothelial cells.

Platelets play a crucial role in the maintenance of hemostasis, as well as in throm-

bosis. Upon activation, platelets release small membrane-bound microparticles (MPs)

containing bioactive proteins and genetic materials from their parental cells that

may be transferred to, and exert potent biological effects in, recipient cells of the

circulatory system. Platelets have been shown to contain an abundant and diverse

array of microRNAs, and platelet-derived MPs are the most abundant microvesicles

in the circulation. Here we demonstrate that human platelets activated with thrombin

preferentially release their miR-223 content in MPs. These MPs can be internalized

by human umbilical vein endothelial cells (HUVEC), leading to the accumulation of

platelet-derived miR-223. Platelet MPs contain functional Argonaute 2 (Ago2)•miR-223

complexes that are capable of regulating expression of a reporter gene in recipi-

ent HUVEC. Moreover, we demonstrate a role for platelet MP-derived miR-223 in

the regulation of 2 endogenous endothelial genes, both at the messenger RNA and

protein levels. Our results support a scenario by which platelet MPs may act as

intercellular carriers of functional Ago2•microRNA complexes that may exert

heterotypic regulation of gene expression in endothelial cells, and possibly other recipient cells of the circulatory system.

(Blood. 2013;122(2):253-261)

Introduction

Platelets are discoid, anucleate cytoplasmic fragments released bybone marrow megakaryocyte precursor cells into the circulationwhere they play a central role in the maintenance of hemostasis, aswell as in thrombosis.1 Platelets are recruited to, and are activatedat, sites of damaged vessel walls or atherosclerotic plaques adjacentto the endothelial lining.

We reported that human platelets contain an abundant anddiverse array of microRNAs2,3 that may be involved in regulatingplatelet messenger RNAs (mRNAs),3 protein synthesis,4 and reac-tivity.5 MicroRNAs are 19- to 24-nucleotide noncoding RNAs6,7

generated by the ribonuclease III Dicer8 and incorporated into effectorArgonaute 2 (Ago2) complexes.9 The biological role of microRNAsis linked mainly to their ability to act in concert and mediate sequence-specific regulation (mainly repression10) of mRNA translation throughrecognition of specific binding sites usually located in the 39untranslated region (UTR). Predicted to regulate;60% of the genesin humans,11 microRNAs may be implicated in the regulation of everycellular process, and changes in their expression and/or function havebeen associated with human genetic diseases.12,13

Whereas the majority of microRNAs are found intracellularly, anumber of microRNAs have also been detected outside of cells, invarious bodyfluids, such as serumor plasma.14CirculatingmicroRNAs

may be found in exosomes,15 shedding vesicles,16 and apoptoticbodies,17 as well as in vesicle-free ribonucleoprotein complexes, inassociation with Ago218 or high-density lipoproteins (HDL).19

Activated platelets may also release microparticles (MPs), smallextracellular vesicles ranging from 0.1 to 1 mm in diameter shedfrom the cytoplasmic membrane. The MPs derived from plateletsare the most abundant cell-derived MP subtype in the circulation20

and may contribute to inflammatory diseases, such as arthritis21

and atherosclerosis.22 In addition to sharing the surface markersof their parental cells, MPs carry a broad variety of cytoplasmiccomponents, including proteins, DNA, and RNA.16 These smalllipid vesicles may act, therefore, as intercellular carriers and deliverbioactive proteins and RNAs to recipient cells, including mRNAs23

and small mRNA regulatory microRNAs.24 MPs may, therefore,play an important role as a cargo of genetic information from onecell type to another across the circulatory system,16 including theendothelial cells that line the inner surface of the vasculature.

The relative abundance and diversity of platelet microRNAs,2,3

the propensity of platelets to release MPs upon activation,19 themicroRNA content of platelet-derived MPs,25 and the relativelyhigh level of circulating microRNAs originating from platelets26

prompted us to investigate the possible intercellular transfer of

Submitted March 23, 2013; accepted April 26, 2013. Prepublished online as

Blood First Edition paper, May 7, 2013; DOI 10.1182/blood-2013-03-492801.

The online version of this article contains a data supplement.

There is an Inside Blood commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2013 by The American Society of Hematology

BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2 253




platelet microRNAs via MPs and assess their capacity for heterotypicregulation of gene expression in recipient endothelial cells.

Methods

Platelet purification, activation, and MP isolation

Platelets were isolated from venous blood, as previously described,3 harvested bycentrifugation at 1000 g for 10 minutes and resuspended at 108 platelets/mL inHEPES-Tyrode buffer (130 mM NaCl, 3 mM KCl, 0.3 mM Na2HPO4, 12 mMNaHCO3, 20 mM HEPES, 5 mM monohydrate D-glucose, 0.5 mM MgCl2,pH 7.4). Platelet activation and MP release were induced upon incubation with0.1 U/mL thrombin (Sigma-Aldrich, St. Louis, MO) for 15 or 60 minutes at37°C with gentle agitation. Platelet activation was stopped by the addition of 20mM final EDTA, and platelets were pelleted by centrifugation at 3,200 g for 10minutes. The supernatantwas centrifugedagain to prepare a platelet-free releasate,whichwas used forMP isolation.MPswere harvested by centrifugation at 20 000g for 90 minutes at 18°C, and were either resuspended in HEPES-Tyrode bufferfor cell coincubations or its RNA extracted by addition of TRIzol (Invitrogen,Carlsbad, CA). The supernatant fraction was collected, snap frozen, and stored at280°C until analyses.

Flow cytometry

Platelets and the MP fractions were analyzed by flow cytometry todetermine their activation status and origin, respectively. Platelets werelabeled with CD62P-R-phycoerythrin and CD41a-anti-allophycocyanin(APC) (BD Biosciences, San Jose, CA), and MPs were labeled withCD41a-APC (BD Biosciences). Approximately 10% of unstimulatedexpressed CD62P at their surface, suggesting that our freshly isolatedplatelets were minimally activated, as compared with ;98% of the plateletsactivated with thrombin (supplementary Figure 1).

MPs (1 mL) were diluted in 100 mL of phosphate buffered saline andincubated in the presence of APC-conjugated mouse IgG (isotype) or APC-conjugated anti-human CD41a (BD Biosciences) for 30 minutes. Then thesamples were diluted to 500 mL in phosphate buffered saline and analyzedcytofluorometrically using a forward-scattered light coupled to a photomultipliertube (PMT) option (forward-scattered PMT) (BD Biosciences) mountedon a Canto II SORP flow cytometer (BD Biosciences). The cytometer wascalibrated before all data acquisitions using BD cytometer setup andtracking beads (BD Biosciences).

Cell culture

HUVEC (Stem Cell Technologies, Vancouver, BC, Canada) were cultured inendothelial growth medium (Lonza, Basel, Switzerland) supplemented withbovine brain extract (Lonza) and maintained at 37°C under 5% CO2. For allexperiments, HUVEC were used between passages 2 to 6. For transfection,5 3 105 cells were transfected by nucleofection on the Nucleofector IIapparatus (Lonza) using the AMAXA HUVEC Nucleofector kit (Lonza).Twenty-four hours later, the culture medium was changed and the MPswere added for up to 48 hours of coincubation.

Production of labeled MP and HUVEC-MP coincubation assays

Isolated human platelets (108 platelets/mL in HEPES-Tyrode buffer)were incubated with 1 mM CellTracker Orange CMTMR (5-[and-6]-[([4-Chloromethyl]Benzoyl)Amino]Tetramethylrhodamine) (Invitrogen) for15 minutes at 37°C in darkness, prior to platelet activation with 0.1 U/mLthrombin. The labeled platelet-derivedMPs were recovered by centrifugation,resuspended in HEPES-Tyrode buffer, and counted by flow cytometry.HUVEC were incubated with fluorescent MPs at a ratio of 1:100 (HUVEC:MPs) for up to 48 hours at 37°C under 5% CO2. MP internalization wasevaluated by confocal microscopy analysis with a spinning disc confocalmicroscope using a363 objective (Quorum Spinning Disc Wave FX, QuorumTechnologies, Guelph, ON, Canada). Up to 24 images, corresponding to asmany 0.5 mm-thick layers, were acquired using the Volocity software

(PerkinElmer, Waltham, MA). Single representative images of the centrallayers are shown.

RNA extraction, microRNA quantification, and gene

expression studies

Total RNA was extracted from platelet-derived MPs and HUVEC using TRIzolreagent (Invitrogen), and from the supernatant fraction using mirVana PARISkit (Ambion, Austin, TX). Reverse transcription reactions were performedwith 1 mg total RNA using HiFlex miSCRIPT RTII kit (Qiagen, Hilden,Germany) after DNase I treatment (Invitrogen). Mature miR-223 and 2selected mRNAs were detected by quantitative PCR (qPCR) using miScriptPrimer Assay kit and SYBR Green (Qiagen). Small nuclear RNAU6 (RNU6)(for miR-223) and glyceraldehyde-3-phosphate dehydrogenase (for mRNAs)were used as reference genes for relative quantitation using the 2^-DDCt

method.27 The sequence of the oligonucleotides used for qPCR quantitationof selected endothelial mRNAs are provided in supplementary Table 1.

Immunoprecipitation and functional assay of

Ago2•microRNA complexes

MPs derived from activated platelets were lysed in RNA immunoprecipitationlysis buffer, and the lysates cleared by centrifugation prior to immunopre-cipitation using protein G-agarose beads (Roche Applied Science, Penzberg,Germany) conjugated with anti-Ago2 antibody (clone 2E12-1C9, Abnova)or isotypic IgG control (anti-FLAG; Sigma-Aldrich), as previously de-scribed.3,28 Ago2-associated miR-223 was isolated by phenol/chloroformextraction and ethanol precipitation, reverse transcribed with the miScript IIRT kit (Qiagen), and analyzed by qPCR using hsa-miR-223 miScriptPrimer Assay (Qiagen).

Ago2×miR-223 function was evaluated in RNA-induced silencing complexactivity assays, as previously described.3

Reporter gene activity assays

Reporter gene activity assays were performed essentially as previouslydescribed.2,29,30 A miR-223 reporter construct was created by inserting asequence complementary to hsa-miR-223 in the Xba1 site of pRL-CMVvector (Promega, Madison, WI), downstream of the Renilla luciferase (Rluc)reporter gene. pRL-CMV-39UTR Ephrin A1 (EFNA1) and pRL-CMV-39UTR F-box/WD repeat-containing protein 7 (FBXW7) constructs wereengineered by amplifying and cloning their 39UTR element downstream ofthe Rluc reporter gene in pRL-CMV vector. All the constructs wereverified by DNA sequencing. The pGL4.51 vector (Promega) expressingFirefly luciferase (Fluc) was used as a normalization control. Both pRL-CMV and pGL4.51 constructs were co-transfected in HUVEC 24 hoursprior to incubation with MPs for up to 48 hours. Rluc and Fluc activitieswere measured with Dual Glo luciferase reagents (Promega) using aluminometer (Dynex Technologies, Chantilly, VA).

Western blot analysis

Protein extracts were analyzed by 10% (wt/vol) sodium dodecyl sulfatepolyacrylamide gel electrophoresis and immunoblotting using anti-Ago2(Abnova, Taipei City, Taiwan), anti-EFNA1 (Abcam, Cambridge, UK), anti-FBXW7 (Invitrogen), and anti-b-actin (AC-40; Sigma-Aldrich) antibodies,followed by enhanced chemiluminescence detection and densitometricanalyses, as previously described.3,29

Blood collection from healthy volunteers (adult Caucasians of both sexesfrom the immediate region of Quebec City) was approved by our institutionalhuman ethics committee. The participants provided their written informedconsent to participate in this study in accordance with the Declaration ofHelsinki, as approved by our institutional human ethics committee.

Results

Activated platelets release MPs that contain miR-223

Flow cytometry analyses of MPs purified from resting or thrombin-activated platelets unveiled a predominant population of MPs,

254 LAFFONT et al BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2




approximately 100 to 400 nm in diameter, which together withplatelet glycoprotein CD41a surface expression (Figure 1A) con-firmed the platelet origin of these MPs.31

The number of MPs released from platelets significantly in-creased by 2.1-fold as early as 15 minutes after stimulation withthrombin (Figure 1B). This increase reached up to 7.3-fold after60 minutes. Knowing that platelets can release microRNAs25,26 andthat microvesicles may contain microRNAs,16 we assessed whether

Figure 1. Activated platelets release MPs that contain miR-223. (A-C) Human

platelets were activated with thrombin (0.1 U/mL) for 15 or 60 minutes, and the MPs

released were isolated by ultracentrifugation. (A) Representative flow cytometry

analyses of the MP population derived from platelets activated with thrombin for

60 minutes. Because of the size of heterogeneity of the MPs,49 fluorescent Sky Blue

microspheres, ranging from 90 nm to 3200 nm in diameter (Spherotech, Lake Forest,

IL), were used to calibrate our flow cytometer and estimate the size of the MPs. The

CD41a1 events were portrayed as forward-scattered light (FSC) and side-scattered light

(SSC) PMT graph using the BD FACSDiva software. (B) MPs were counted by flow

cytometry by an FSC coupled to a PMT. Results are expressed as the mean

(6 standard error of the mean [SEM]) fold changes vs unstimulated platelets, used

as a reference (n 5 3 experiments). (C) The MP and supernatant fractions of

thrombin-activated platelets were isolated and analyzed for their content in miR-223

by qPCR. Results were normalized by the 2^-DDCt method, using RNU6 as a

reference,27 and expressed as the mean (6SEM) fold changes vs unstimulated

platelets (n 5 3 experiments). Similar results were obtained by monitoring 4

additional microRNAs (data not shown). *P , .05 vs baseline (Student t test). SSC,

side-scattered light.

Figure 2. Platelet-derived MPs contain functional Ago2•miR-223 effector

complexes. (A-C) Human platelets (PLT) were activated with thrombin (0.1 U/mL)

for 60 minutes, and the MP fraction was isolated by ultracentrifugation. Unstimulated

platelets served as the control.3 (A) The presence of Ago2 was assessed by

immunoblot (IB) analysis using an anti-Ago2 antibody. (B) Protein extracts derived

from the MP fraction were subjected to immunoprecipitation using anti-Ago2 antibody,

followed by quantitative miR-223 detection by qPCR. Results were normalized by the

2^-DDCt method, using RNU6 as a reference,27 and expressed as a percentage of

the input (mean 6 SEM; n 5 4 experiments). *P , .05 vs normal isotypic IgG, which

was used as an IP control (Student t test). (C) The supernatant (S100) (50 mg proteins)

fraction of protein extracts derived from PLT or the MP fraction of thrombin-activated

platelets were subjected to RNA target cleavage assays, using a 59 end, 32P-labeled

miR-223 RNA sensor. RNA was isolated by phenol/chloroform extraction and ethanol

precipitation, separated by denaturing 8% polyacrylamide gel electrophoresis PAGE/7

M urea and visualized by autoradiography (n 5 2 experiments). *The 39-nucleotide

RNA product expected from Ago2-mediated endonucleolytic cleavage of the sensor. -,

uncleaved 32P-labeled miR-223 RNA sensor.

BLOOD, 11 JULY 2013 x VOLUME 122, NUMBER 2 ENDOTHELIAL GENE REGULATION BY PLATELET miR-223 255




activated platelets release miR-223 (one of the most abundant plateletmicroRNA)2,3 either directly into the supernatant or through MPs.Little or no changes were observed in supernatant microRNA levels(Figure 1C). However, we observed a significant ;60-fold increasein MP miR-223 levels 60 minutes after platelet activation withthrombin, as compared with baseline (Figure 1C), suggesting thatthrombin-activated platelets may preferentially release miR-223through MPs.

Platelet-derived MPs contain functional Ago2•miR-223

effector complexes

MicroRNAs are known to guide effector complexes containing Agoproteins, such as Ago2,9 for the regulation of specific mRNAs throughtranslational repression, mRNA destabilization, or a combinationof the these.11 Immunoblot analysis of platelet-derived MP proteinextracts revealed the presence of Ago2 proteins (Figure 2A, lane 2),which together with the presence of MP miR-223 prompted us toverify if Ago2 and miR-223 exist in MPs in the form of a complex.

With the aim to subsequently study the role of platelet-derivedMP microRNAs in regulating endothelial cell gene expression, weselected a microRNA that is highly abundant in platelets while beingexpressed at very low levels in endothelial cells (ie, miR-223).3,24,25,32

Ago2 immunoprecipitation followed by qPCR detection of miR-223

unveiled the existence of an Ago2×miR-223 complex in MPs releasedfrom thrombin-activated platelets (Figure 2B). The levels of miR-223associated with MP Ago2 proteins were enriched by more than300-fold compared with an isotypic control IgG (anti-FLAG anti-body) (supplementary Figure 2).

To determine whether MP Ago2×miR-223 complexes are func-tional, we performed RNA-induced silencing complex activity assaysin which MP or platelet protein extracts were incubated inthe presence of a 32P-labeled RNA sensor bearing a sequencecomplementary to hsa-miR-223. We observed the cleavage of theRNA sensor into a 39-nucleotide RNA product (Figure 2C), sup-porting the functionality of Ago2×miR-223 complexes present inMPs released from activated platelets. Together, these resultssuggest that platelet-derived MPs can participate to intercellularcommunication by delivering functional Ago2×microRNA com-plexes to recipient cells.

Platelet-derived MPs are internalized by HUVEC

First, we used confocal fluorescence microscopy to determine ifplatelet-derived MPs could be internalized by HUVEC. We observedthe presence of well-defined punctate signals in the cytoplasm ofHUVEC exposed to fluorescently labeled platelet-derived MPs for1 hour (Figure 3A, upper panel, second from left), which is consistent

Figure 3. Platelet-derived MPs are internalized by

HUVEC. (A-B) HUVEC were incubated for the indicated

periods of time (t), with fluorescent-labeled MPs at an

HUVEC:MPs ratio of 1:100, and an MP uptake was

visualized by confocal microscopy (363 objective)

(upper panels). Cell morphology was visualized using

differential interference contrast (DIC) (lower panels).

(B) HUVEC were incubated with fluorescently labeled

MPs for 3 hours, and MP uptake was visualized by

confocal microscopy after a 24- or 48-hour washout

period (upper panels). The images are representative of

3 independent experiments.





with the internalization of platelet-derived MPs by HUVEC. Thefluorescent signal persisted for at least 24 hours, raising the issue asto whether this is due to the continuous uptake of MPs or the stabilityof MPs in HUVEC. To verify that possibility, we coincubatedHUVEC with labeled MPs for 3 hours to allow MP internalizationbefore washing the cells, changing the medium, and prolonging theincubation period to 48 hours. We observed that the cytoplasmicpunctate staining of HUVEC, conferred by the fluorescent MPs,persisted for 48 hours in the absence of exogenous MPs (Figure 3B,upper panels). We noticed that the fluorescence seemed to havediffused over time (Figures 3A-B), which is in accordance withthe release of the MP content in the HUVEC cytoplasm.

Platelet-derived MPs can deliver functional Ago2•miR-223

effector complexes to HUVEC

Knowing that platelet-derived MPs contain an appreciable amountof micoRNAs,24,25 we wanted to determine if MPs could delivertheir microRNA content to HUVEC after their internalization.Focusing on miR-223, which is highly abundant in platelets3 butweakly expressed in HUVEC,32 we observed a 22-fold increase inHUVEC miR-223 levels as early as 1 hour after incubation withplatelet-derived MPs (Figure 4A). This significant enrichment in

HUVEC miR-223 levels persisted for up to 48 hours, comparedwith HUVEC incubated without MPs. The marked increase andpersistence of elevated miR-223 levels in HUVEC may providethe conditions and time window necessary for platelet-derivedAgo2×miR-223 to regulate HUVEC gene expression.

To verify this, we transiently transfected HUVEC with a reportergene construct, in which Rluc was placed under the control of amiR-223 binding site, prior to incubation with platelet-derived MPs.We observed a significant 44% decrease in HUVEC reporter geneactivity induced by coincubation with MPs (Figure 4B). Theseresults indicate that platelet MP-derived Ago2×miR-223 complexescan target specific mRNAs and exert gene regulatory effects inrecipient endothelial cells.

Platelet MP-derived miR-223 can regulate HUVEC gene

expression at the mRNA level

To investigate whether MP miR-223 derived from platelets canregulate endogenous HUVEC gene expression, we searched for po-tential endothelial mRNA targets of platelet miR-223, and identified2 mRNA candidates: FBXW7, an onco-suppressor protein andvalidated mRNA target of miR-22333; and EFNA1, a glycosylphos-phatidyl inositol-anchored receptor tyrosine kinase ligand.34 FBXW7and EFNA1 mRNAs harbor 4 and 1 conserved miR-223 binding sitesin their 39UTR, respectively (supplementary Figure 3).35

Using a reporter gene-based assay in HUVEC, we observed asignificant 47% and 31% downregulation of Rluc reporter geneactivity induced upon coincubation with MPs and conferred by the39UTR of FBXW7 (Figure 5A, left panel) and EFNA1 (Figure 5A,right panel) mRNAs, respectively. These results indicate that platelet-derived MPs can modulate gene expression through regulatoryelements located in the 39UTR of 2 selected endothelial mRNAs.

We observed a significant downregulation of endogenous FBXW7and EFNA1 mRNA levels that reached a maximum of 51%(Figure 5B, left panel) and 28% (Figure 5B, right panel), respectively,within 6 hours of exposure to MPs. To ascertain that these effects aremediated by platelet MP-derived miR-223, we transiently transfectedHUVEC, with a vector expressing a mRNA bearing a sequence com-plementary to miR-223 and acting as a miR-223 sponge, prior toincubation with MPs. As shown in Figure 5C, the miR-223 spongeneutralized the down-regulatory effects induced by platelet MPson the endogenous FBXW7 (Figure 5C, left panel) and EFNA1(Figure 5C, right panel) mRNA levels, and even enhanced EFNA1mRNA levels above baseline. This latter observation may be due tothe sequestration of endothelial miR-223, which may regulate endog-enous FBXW7 expression to a greater extent than EFNA1. Thesefindings support a role for platelet MP-derived miR-223 in regulatingFBXW7 and EFNA1 expression at the mRNA level.

Platelet MP-derived miR-223 can regulate HUVEC gene

expression at the protein level

The mRNA regulatory effects of platelet MP-derived miR-223 couldbe translated at the protein levels, as we observed a marked decreasein HUVEC FBXW7 protein levels as early as 18 hours after ex-posure to platelet-derived MPs (Figure 6A, left panel). This effectpersisted for up to 48 hours, suggesting that platelet MP-derivedmiR-223 may influence endothelial gene expression for extendedperiods of time. HUVEC EFNA1 protein levels were also decreasedby platelet-derived MPs, but only after 96 hours of coincubation(Figure 6A, right panel), which is probably due to a slower turnoverof cellular EFNA1 proteins compared with FBXW7.

Figure 4. Platelet-derived MPs can deliver functional Ago2•microRNA effector

complexes to HUVEC. (A) HUVEC were incubated with platelet-derived MPs for up

to 48 hours, and miR-223 accumulation in HUVEC was quantitated by qPCR.

Results were normalized by the 2^-DDCt method, using RNU6 as a reference,27 and

expressed as the mean (6SEM) fold changes vs baseline (n 5 5 experiments).

(B) HUVEC transiently expressing a Rluc reporter gene, harboring a binding site

complementary to miR-223 (pRL-CMV-BS miR-223), were incubated (or not; control)

with MPs derived from thrombin-activated platelets for 48 hours prior to luciferase

activity measurements. Results were normalized on Fluc activity, and expressed as

mean (6SEM) percentage of control (n 5 4 experiments). *P , .05; **P , .01 vs

baseline or control (Student t test). pRL-CMV, Renilla luciferase–cytomegalovirus

(CMV) plasmid; BS miR-223, Binding site for miR-223.





Together, these results are consistent with a scenario by whichactivated platelets may deliver, through the release of MPs, mRNAregulatory Ago2×microRNA complexes to other cells of the cardio-vascular system and regulate expression of endogenous genes inrecipient cells, such as endothelial cells (Figure 6B).

Discussion

Although recent studies tend to support a mRNA regulatory rolefor platelet microRNAs4 and Ago2×microRNA complexes3 in plateletbiology,4,5 the definite proof is still being sought, because of thechallenge and intrinsic limitations associated with working withprimary human platelets, including their relative refractoriness totransfection.3 The propensity of platelets to release MPs upon acti-vation,21 the microRNA content of circulating MPs derived fromplatelets,25 and the capacity of MPs to transfer their content torecipient cells16 made it tempting to investigate a possible roleof platelet microRNAs outside of platelets.

The pool of circulating microRNAs is composed of vesicle-associated microRNAs, as well as protein-bound microRNAs thatoriginate from different cell types, including platelets,26 fromwhich the most abundant MP subtype in the circulation derive.20 Inthe present study, we found that thrombin-activated platelets releasea majority of their miR-223 content in MPs, whereas minute amountsseem to be released directly into the supernatant. These findings aresupported by Diehl et al,24 who reported that a prominent amountof plasma microRNAs is associated to MPs. The increase in MPmiR-223 levels was proportionally superior to, and correlated with,the number of MPs released by thrombin-activated platelets (;60-foldversus 7.3-fold). These results suggest that thrombin may induce

the release of platelet MPs enriched in miR-223, compared withMPs produced spontaneously by resting platelets. However, MPsmay not have the monopoly of intercellular microRNA transfer, ascirculating microRNAs may also be delivered to target cells viaHDL.19 Vickers et al19 reported that miR-223 complexed withHDL can be efficiently transferred to cocultured hepatocytes andinfluence the expression of miR-223 targets in recipient cells. Ourresults suggest that activated platelets may not contribute toa significant extent to the pool of vesicle-free forms of circulatingmicroRNAs that are either associated with Ago218 or HDL.19

Platelets may also release microvesicles smaller than MPs, calledexosomes (40 to 100 nm in diameter),36 that may not have sedimentedwith MPs upon centrifugation at 20 000 g. Although miR-223 ofexosomal origin could have contributed to the miR-223 signaldetected in the supernatant fraction, its overall contribution to thepool of miR-223 released from activated platelets is almost negligiblecompared with MPs. The caveat has to be taken into account thatthe profile and mode of microRNA release from platelets may dependon the nature of the agonist(s), stimulatory, and/or shear conditions.

Released upon activation or apoptosis of almost every blood celltype,37 MPs have been associated with a variety of pathologies,including arthritis21 and atherosclerosis,22 as well as tumor progres-sion, angiogenesis, and metastasis.38 This may be explained by theproperties of MPs to enhance vascular permeability,39 to promoteinflammation,21,31 and to act as a procoagulant.40 Although cytokinesmay account for most of these biological effects,22,37 it would not beprudent to dismiss a possible role for bioactive molecules, other thancytokines such as microRNAs, in MP-mediated effects in thecirculatory system.

MPs and other vesicles, such as exosomes and even apoptoticbodies, can act as carriers and mediate the horizontal transfer ofproteins and RNAs between cells. For instance, mRNAs contained

Figure 5. Platelet MP-derived miR-223 can regulate

HUVEC gene expression at the mRNA level. (A)

HUVEC transiently expressing a Rluc reporter gene,

harboring the 39UTR of FBXW7 (pRL-CMV-39UTR

FBXW7) (left panel) or EFNA1 mRNA (pRL-CMV-

39UTR EFNA1) (right panel), were incubated (or not;

control) with MPs derived from thrombin-activated

platelets for 48 hours prior to luciferase activity mea-

surements. Results were normalized on Fluc activity,

and expressed as mean (6SEM) percentage of control

(n 5 3 experiments). (B-C) HUVEC transiently express-

ing a miR-223 sponge (pRL-CMV-BS miR-223 vector;

n = 3 experiments) (C), or not (n = 5 experiments) (B),

were incubated with platelet-derived MPs for up to 48

hours, and FBXW7 (left panels) and EFNA1 (right panels)

mRNA levels were quantitated by qPCR. Results were

normalized by the 2^-DDCt method, using glyceraldehyde-

3-phosphate dehydrogenase mRNA as a reference, and

expressed as mean (6SEM) fold changes vs baseline.

*P , .05; **P , .01; ***P , .0001 vs control or baseline

(Student t test).





in murine mast cell-derived exosomes may be transferred to humanmast cells and induce mouse protein expression.15 Platelet-likeparticles were also shown to transfer their mRNA content torecipient vascular cells.23 Similarly, Zernecke et al17 demonstratedthat miR-126 derived from endothelial apoptotic bodies could beefficiently delivered to neighboring endothelial cells and have anatheroprotective role in the mouse. By demonstrating a role forplatelet MPs in miR-223 delivery to recipient cells of endothelialorigin where it can modulate expression of endogenous genes, ourstudy highlights the relative complexity, efficiency, and functional

importance of intercellular communications across the circulatorysystem. These communications are even more complex, consideringthe diversity of platelet microRNA sequences,2 as well as the numberof cell types capable of exchange (ie, releasing, internalizing, andintegrating, genetic materials or information) through differentmodes of intercellular transfer. In that context, a microvesicularcarrier harboring specific surface proteins may confer a certaindegree of specificity and provide a means of delivering plateletmicroRNAs to specific cells. In view of our findings, it may bereasonable to speculate that MPs released by activated platelets may

Figure 6. Platelet MP-derived miR-223 can regulate

HUVEC gene expression at the protein level. (A)

Protein extracts prepared from HUVEC, incubated or

not with MPs derived from thrombin-activated platelets

for up to 48 or 96 hours, were analyzed by immu-

noblotting (IB) using anti-F-box and WD-40 domain

protein 7 (anti-FBXW7) (left upper panel; n 5 3

experiments), anti-EFNA1 (right upper panel; n 5 1

experiment) or anti-actin (lower panels) antibody, which

was used as a loading control. The data were analyzed

by densitometry and expressed as a percentage of

control. (B) Proposed model for the intercellular transfer

of functional Ago2•microRNA complexes between acti-

vated platelets and endothelial cells through the release

of MPs. RNA pol II, RNA polymerase II.





communicate this information, and modulate, indeed adapt, theresponsiveness of the vasculature, accordingly. The abundance ofcirculating MPs may underlie their relative importance in mediatingnormal regulatory functions and cell-to-cell communications/coordinations, whose dysfunction may contribute to pathophys-iological conditions.21

MPs of various origins contain microRNAs and can be internalizedby recipient cells.24,25,41 In this study, we report the internalizationby HUVEC of platelet MP-derived miR-223, which maintains itsfunctionality and regulates endothelial expression of 2 of itsvalidated mRNA targets (ie, FBXW7 and EFNA1). EFNA1 is amarker of liver cancer,42 whereas FBXW7 was unveiled as ageneral tumor suppressor in human cancer.43 Both genes are (down)regulated by miR-223 at the mRNA and protein levels. Mechanis-tically, MP-derived miR-223 may regulate FBXW7 and EFNA1gene expression, either through mRNA destabilization, inhibition ofmRNA translation initiation, or both.11 The fact that downregulationof mRNA levels preceded that of protein levels militates in favor ofmiR-223-mediated destabilization of these 2 endothelial mRNAs.

Although our results support the involvement of platelet-derivedAgo2×microRNA complexes in regulating endothelial mRNA trans-lation, we cannot exclude the possibility that MP microRNAs, freeof Ago2 proteins, may integrate the microRNA machinery of endo-thelial cells to mediate their mRNA regulatory effects. In addition,whether platelet-derived microRNAs can mediate epigenetic effectsin recipient cells should also be considered.

MiR-223 may be implicated in cell proliferation,44 in osteoclast,erythroid or megakaryocyte differentiation,45,46 and in cancer.47

Therefore, the delivery of platelet miR-223 to other cell types viaMPs may have important implications that can only be magnified bythe plurality of microRNA targets and the diversity of the microRNApopulation released through MPs. Such intercellular exchanges ofmicroRNAs mediated by platelet-derived MPs may contribute to thegene expression programming of recipient cells and to the condi-tioning of the circulatory system under specific health and diseaseconditions associated with platelet activation.

The in vivo relevance of this process has gained support froma recent study performed by Gidlof et al,48 in which the authorsreported the (down)regulation of intercellular adhesion molecule 1

gene expression in cultured human microvascular endothelial cell–1cells exposed to a microRNA (ie, miR-320b) that is released uponplatelet activation and is found to be depleted in platelet-containing thrombi aspirated from patients with ST-elevationmyocardial infarction. The process that we documented may bemanipulated and eventually lead to the development of newtherapeutic modalities aimed to improve the circulatory functionof cardiovascular patients.

Acknowledgments

A.-C.D. was supported by a studentship from the Canadian ArthritisNetwork. E.B. is a New Investigator of the Canadian ArthritisNetwork and Junior 1 Scholar from the Fonds de recherche duQuebec – Sante.

This work was supported by a grant from the Canadian BloodServices/Canadian Institutes of Health Research (CIHR) BloodUtilization and Conservation Initiative via Health Canada (286777)(P.P.) and by a grant from The Arthritis Society/CIHR (244472)(E.B.).

Authorship

Contribution: P.P. conceived and coordinated the project; B.L.led the project; B.L, E.B., and P.P. designed and planned theexperiments; B.L., A.C., H.P., A.-C.D. and N.C. performed theexperiments and analyzed the data; B.L., A.C., H.P., A.-C.D.,N.C., E.B., and P.P. commented on and edited the manuscript;and B.L and P.P. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Patrick Provost, CHUQ Research Center/CHUL,2705 Blvd Laurier, Room T1-49, Quebec, QC G1V 4G2 Canada;e-mail: [email protected].

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