The C2238/aANP Variant Is a Negative Modulator of Both Viability and Function of Coronary Artery ...

12 Pages·2014·1.14 MB·English

The C2238/aANP Variant Is a Negative Modulator of Both
Viability and Function of Coronary Artery Smooth Muscle
Cells
Speranza Rubattu1,2*, Simona Marchitti1, Franca Bianchi1, Sara Di Castro1, Rosita Stanzione1,
Maria Cotugno1, Cristina Bozzao2, Sebastiano Sciarretta1, Massimo Volpe1,2
1IRCCSNeuromed,Pozzilli(Isernia),Italy,2DepartmentofClinicalandMolecularMedicine,SchoolofMedicineandPsychology,UniversitySapienzaofRome,OspedaleS.
Andrea,Rome,Italy
Abstract
Background:Abnormalitiesofvascularsmoothmusclecells(VSMCs)contributetodevelopmentofvasculardisease.Atrial
natriureticpeptide(ANP)exertsimportanteffectsonVSMCs.AcommonANPmolecularvariant(T2238C/aANP)hasrecently
emerged asanovel vascular riskfactor.
Objectives:We aimed atidentifying effects of CC2238/aANP on viability, migration and motility incoronary artery SMCs,
and theunderlying signalingpathways.
MethodsandResults:CellswereexposedtoeitherTT2238/aANPorCC2238/aANP.Attheendoftreatment,cellviability,
migration and motility were evaluated, along with changes in oxidative stress pathway (ROS levels, NADPH and eNOS
expression), onAkt phosphorylation and miR21 expression levels. CC2238/aANP reduced cell vitality,increased apoptosis
andnecrosis,increasedoxidativestresslevels,suppressedmiR21expressionalongwithconsistentchangesofitsmolecular
targets (PDCD4, PTEN, Bcl2) and of phosphorylated Akt levels. As a result of increased oxidative stress, CC2238/aANP
markedlystimulatedcellmigrationandincreasedcellcontraction.NPR-CgenesilencingwithspecificsiRNAsrestoredcell
viability,miR21expression,andreducedoxidativestressinducedbyCC2238/aANP.ThecAMP/PKA/CREBpathway,drivenby
NPR-C activation, significantly contributed to both miR21 and phosphoAkt reduction upon CC2238/aANP. miR21
overexpression by mimic-hsa-miR21rescued the cellulardamagedependent onCC2238/aANP.
Conclusions:CC2238/aANPnegativelymodulatesviabilitythroughNPR-C/cAMP/PKA/CREB/miR21signalingpathway,andit
augmentsoxidativestressleadingtoincreasedmigratoryandvasoconstrictoreffectsincoronaryarterySMCs.Thesenovel
findingsfurthersupportadamagingroleofthiscommonaANPvariantonvesselwallanditspotentialcontributiontoacute
coronaryevents.
Citation:RubattuS,MarchittiS,BianchiF,DiCastroS,StanzioneR,etal.(2014)TheC2238/aANPVariantIsaNegativeModulatorofBothViabilityandFunctionof
CoronaryArterySmoothMuscleCells.PLoSONE9(11):e113108.doi:10.1371/journal.pone.0113108
Editor:JunmingYue,TheUniversityofTennesseeHealthScienceCenter,UnitedStatesofAmerica
ReceivedJune28,2014;AcceptedOctober15,2014;PublishedNovember17,2014
Copyright:(cid:2)2014Rubattuetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaperandits
SupportingInformationfiles.
Funding:Thisworkwassupportedbyagrant(RicercaCorrente)fromtheItalianMinistryofHealthtoMVandSR;bythe5%granttoMVandSR.Thefunderhad
noroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected]
Introduction involvedintheseeffectssincemiRNA-21wasshowntocontribute
totheantiproliferative effectof ANPin human aorticSMCs [3].
Atrial natriuretic peptide (ANP) is a cardiovascular hormone
VSMCsplayarelevantroleinvesselphysiologyanddisease[4].
which exerts several beneficial properties on both cardiovascular
In particular, based on their ability to adapt to various stimuli,
hemodynamic and structure [1]. Its vasorelaxant, diuretic and
theycanparticipatetoeitherthevascularrepairprocessortothe
natriureticeffectsaremediatedbythemembrane-boundguanylyl
vasculardiseaseconditionsuchasatheroscleroticplaqueformation
cyclase type A receptor (GC-A) through an increase of cyclic
[5].Importantly,VSMCsarealsorequiredtomaintainstabilityof
guanylate monophosphate (cGMP) levels. On the other hand,
atherosclerotic plaques [6]. These evidences suggest that, by
ANP binding to natriuretic peptide type C (NPR-C) receptor is
regulating VSMC biological functions, ANP may contribute to
known to mediate its clearance [1]. ANP exerts its vasodilatory
bothphysiological and pathologicalvascular processes.
property by inducing vascular smooth muscle cell (VSMC)
The common T2238C molecular variant of human aANP is
relaxation [1]. Previous evidence indicates that ANP can also
emerging as a novel cardiovascular risk factor for its ability to
reduce VSMC proliferation [2]. Notably, microRNAs may be
increase the risk of cardiovascular events both in coronary artery
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T2238C/aANPVariantandCoronaryArterySmoothMuscleCells
diseasepatientsandinthegeneralpopulation[7–12],alongwitha Cellmigrationassay. Totestcellmigration,CASMCswere
significant impairment of endothelium-dependent vasodilation seeded in 24-well plates (86104 cells/well), cultured in their
[13]. These negative effects result from deregulated activation of medium for 24hrs, subsequently starved and stimulated with
NPR-C byCC2238/aANPvariant [13]. eitherTT2238-orCC2238/aANPformfor12hrsinthepresence
The impact of CC2238/aANP on VSMCs has not been of10%FCS.Attheendofstimulation,cellswereresuspendedin
exploredyet.Sinceitsunderstandingmayintegrateknowledgeon their medium, starved and seeded in Boyden chambers (BD
mechanisms of vascular disease promotion dependent on this Biosciences, San Jose, California, USA) to test migration
common aANP molecular variant, we performed the present (chemotaxis). In fact, following a 24-hrs period of incubation,
studies to explore: 1) the effects of CC2238/aANP on coronary cells migrated from the upper part of the chamber to the lower
artery SMC (CASMC) viability, migration and motility; 2) the filterwerefixedbyDiffQuick(DadeBehring,Paris,France)and
pathways underlying alterations of viability and function depen- visualized by microscopy. To test the involvement of both ROS
dentonCC2238/aANPinCASMCs;3)theimplicationofNPR- and cAMP levels in modulating the migratory property of
C driven cAMP/PKA/CREB pathway in modulating cellular CASMCs upon CC2238/aANP, separate sets of experiments
viability through phosphoAkt and miR21 regulation in the were perfomed in the presence of apocynin (200 mmol/l, Sigma)
presence ofCC2238/aANP. andof forskolin (FSK,10mM,Sigma).
Collagen gel lattice contraction assay. To test cell
Materials and Methods contraction, a commercially available kit assay was used (Cell
Biolabs,SanDiego,CA,USA).Twopartsofcellsweremixedwith
1. Effects of exposure to TT2238 and CC2238/aANP in eightpartsofcollagengellatticemixtureandtheywereplatedfor
CASMCs 1 hr at 37uC. Thereafter, 1 ml of medium was added and
Commercially available CASMCs (human coronary artery incubatedfor2days.Next,thegelswerereleasedfromthesidesof
smooth muscle cells obtained from normal donors, Cat. No CC- the wells and full release was allowed for 24hrs. During the last
2583), purchased from Lonza (Walkersville, MD, USA), were 12hrsofreleaseeither1029MTT2238-orCC2238/aANPwas
grown in Smooth Muscle Growth Medium-2 (SmgM-2). They added to cells. After completion of incubation, the area of gel
were used within the 5th passage and at 70% confluence for the lattice was taken with Image J software, and the relative lattice
following setsofexperiments afteranovernightexposure (12 hrs) area was obtained by dividing the area of the well 24hrs after
to either TT2238/aANP (wild type) or CC2238/aANP (variant) detachmentbytheinitialareaofthewell.Totesttheinvolvement
at 1029M concentration, as previously reported for endothelial of both ROS and cAMP levels in modulating cell motility upon
cells(ECs)[13].Thisconcentrationhasbeenusedinourstudiesto CC2238/aANP, separate sets of experiments were performed in
bettermimicthephysiologicalconditionofvascularcellsexposed thepresence ofapocynin (200 mmol/l)and ofFSK(10 mM).
to both circulating and endogenous aANP. Once exposure to
eitheraANPformwascompleted,variablesdescribedbelowwere 2.Investigationof thesignalinginvolved inthe negative
assessed 24hrs later. From three to six experiments were effects of CC2238/aANP in CASMCs
performed foreach of thefollowing studies. From three to six experiments were performed for each of the
Cell viability assessment by trypan blue assay. Cells following studies, unlessotherwiseindicated.
were seeded in 60-mm well plates (26105 cells/well), cultured in Cyclic adenylate monophosphate (cAMP) and cyclic
theirmediumfor24hrs,andsubsequentlystimulatedwitheither guanylate monophosphate (cGMP) levels
TT2238- or CC2238/aANP for 12hrs in the presence of 10% measurement. For this purpose, CASMCs were seeded in
fetal bovine serum (FBS). Twenty-four hrs later, viable and dead six-wellplates(26105cells/well)andculturedinSmgM-2medium
cellswerecountedusingtheTrypanblueexclusionmethodunder for24hrs.Atthistime,theywerefirststarvedfor2 hrsandthen
an opticalmicroscope, as previouslydescribed [13]. pretreated for 30min. with 0.1mmol/L isobutylmethylxanthine
Cell viability assessment by Annexin V-PI (IBMX, Sigma) before stimulation with the conditioned medium
staining. Counting of apoptotic cells was performed by flow containingIBMX0.1 mMandeitherTT2238orCC2238/aANP
cytometry analysis (FACS) using Annexin V-Fitc and Propidium at1029Mconcentrationfor30min.,basedonourowndata[13]
Iodure (PI) staining (ImmunoStep, Salamanca, Spain). For this and on previously published protocols [14]. cGMP and cAMP
purpose, 24hrs after completion of overnight exposure to either measurementswereperformedwithspecificenzymeimmunoassay
aANPform,CASMCswereharvestedbyincubationwith1 mlof Biotrak(EIA)System(Amersham,Piscataway,NewJersey,USA),
trypsin/EDTA (Lonza) for 3 min at 37uC. Trypsinization was following the manufacturer’s instructions (number of experi-
stopped by addition of medium and the suspension was ments=12). To better assess guanylyl cyclase activity following
centrifuged at 1200rpm for 5 min. at 4uC. Each pellet was bindingofbothaANPformswithGC-A(natriureticpeptidetype
washedwithcoldphosphatebufferedsaline(PBS1x).Then,tubes A receptor, NPR-A), a dose-response curve was performed by
were vortexed thoroughly and centrifuged again as before. Cells usingarangeofconcentrationsincludedbetween10211and1026
were gently resuspended and vortexed in binding buffer at a M of each peptide. Furthermore, to support the involvement of
concentration of 36106 cells/ml. Then, 100ml of cell suspension NPR-C, rather than NPR-A, in mediating deleterious effects of
was added to 5 ml of Annexin V-Fitc and 10ml of PI. Samples CC2238/aANP,weassesseditsimpactoncellviability(bytrypan
weremixedfor15min.inthedarkat4uCand400mlofPBS1x blue) in the presence of 500nM anantin (NPR-A antagonist that
was added to the solution. Ten thousand cells were analyzed by blocks cGMP release[15]).
FACSonaBDAccuriC6flowcytometertocountapoptoticcells. Analysis of miR21 expression. At the end of exposure to
Both negative control (untreated cells) and positive control (cells either aANP form, microRNA from cells was immediately
treated withhydrogen peroxide)were included intheanalysis. obtainedusingmirVanamiRNAisolationkit(AppliedBiosystems,
Reactive oxygen species (ROS) levels CA,USA).TheRTreactionformiR21andforU75-controlmiR
assessment. Oxidative stress was evaluated 24hrs after com- wasperformedusingTaqManMicroRNAReverseTranscription
pletion of exposure to either aANP form by applying the DCHF Kit (Applied Biosystems, Milan, Italy). Two ng/mL of RNA, 1X
procedure, aspreviously described[13]. stem-loop RT primer, 3.33 U/mL reverse transcriptase, 0.25 U/
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T2238C/aANPVariantandCoronaryArterySmoothMuscleCells
mL RNase inhibitor, 0.25mM dNTPs, and 1X reaction buffer densitometrically. They were finally normalized using b-actin
were run in a total reaction volume of 15mL and incubated at levels.
16uC for 30min, 42uC for 30min, and 85uC for 5 min in a
thermal cycler. 0.8mL of the RT reaction was combined with 3. Impact of NPR-C gene silencing in CASMCs in the
0.5 mL of a specific TaqMan MicroRNA Assay (U75-control, presence of CC2238/aANP
miR-21, 20X; forward primer, reverse primer, and probe) and
NPR-C gene silencing was performed once cells had reached
5 mL of TaqMan Universal PCR Master Mix in a 10mL final
70%confluence.Atthistime,theywerewashedwithPBS1x,and
volume. Real-time (RT)-PCR was performed using an Applied
OPTI-MEM-reduced serum medium (Invitrogen, Milan, Italy)
Biosystems 7900HT Fast Real-Time PCR System with cycling
was added to the cells. Two NPR-C specific small interfering
conditions of 95uC for 10min followed by 95uC for 15sec and
RNAs (Mission siRNA, Sigma) and a nucleic acid transferring
60uCfor60secforatotalof40cycles.EachTaqManassaywas
agent (RNA I Max lipofectamine, Invitrogen) were incubated in
run in triplicate. Results were expressed as relative levels of each
OPTI-MEM reduced serum medium for 20min. at room
microRNA comparing different treatments.
temperature to form a siRNA-lipofectamine complex. The
A separate set of studies tested the role of cGMP dependent
siRNA-lipofectamine complex-containing medium was added to
kinase (cGK) in mediating the 50% miR21 reduction dependent
thecellstoa finalsiRNAconcentrationof50nM.Fivehrslater,
on wild type aANP. We used cGK inhibitor (Rp-8-Br-PET-
the complex-containing medium was replaced with SmgM-2
cGMPS, Sigma) at concentration of 25mM for 30min. followed
supplementedwith10%FBS.Cellstransfectedwithlipofectamine
by overnight exposure towildtype aANP.
and nonsense siRNA (Sigma Aldrich) were used as control.
WesternblotanalysisofAkt,NADPH(gp91phoxsubunit),
Twenty-four hrs later, both silenced and not silenced cells were
eNOS,miR21targets(PDCD4,PTEN,Bcl2). To determine
exposedtoCC2238/aANPfor12hrsandsubsequentlyanalyzed
protein expression levels 24hrs after completion of exposure to
for: cell viability, eNOS and NADPH protein expression levels,
either TT2238- or CC2238/aANP, samples were lysed in 1:10
ROS levels, cell migration, cell contraction, miR21 and related
weight/volume in lysis buffer (50mM NaCl, 100mM Tris-HCl
targets (PTEN, PDCD4, Bcl2) expression, Akt phosphorylation
pH7.4,1 mMEDTA,1%SDS)supplementedwithproteaseand
levels. Sixexperiments wereperformed forthese studies.
phosphataseinhibitors(Sigma).Sampleswereincubatedonicefor
15min. and centrifuged at 13000rpm for 15min. Protein
4. Impact of miR21 overexpression in CASMCs in the
concentration was determined by DC Protein Assay Kit (Bio-
Rad, Hercules, CA, USA) following manufacturer’s instructions. presence of CC2238/aANP
Proteinlysateswereboiledat95uCfor10min.inpresenceofgel Cellswereusedat70%confluence.Mimichsa-miR21(Mission
loading buffer (Bio-Rad). 50mg of each sample were loaded on microRNA,Sigma)andRNAIMaxlipofectaminewereincubated
10% SDS-polyacrylamide gel and run for 120min. at 100V. in OPTI-MEM reduced serum for 20min. at room temperature
Proteins were then transferred to polyvinylidene difluoride toformamimic-lipofectaminecomplex.Thecomplexcontaining
membranes (Amersham) using the Trans-Blot Turbo Transfer medium was added to the cells to reach a final mimic miR21
System(Bio-Rad).Nonspecificbindingsiteswereblockedatroom concentration of 100nM. Five hrs later the complex containing
temperature for 1 hr in 5% skim-milk in Tris-buffered saline medium was replaced with SmGM-2 supplemented with 10%
buffer +0.1% Tween 20 (Sigma) (TBS-T). Membranes were FBS. Cells transfected with lipofectamine and Mission miRNA
incubatedat4uCovernightwithprimaryantibodiesdilutedin5% negative control (Sigma) were used as control. Twenty-four hrs
bovine serum albumine (BSA) in TBS-T. The following primary after transfection cells overexpressing miR21 were exposed to
antibodieswereused:1:200rabbitpolyclonalanti-proteinkinaseB CC2238/aANP for 12hrs and used, 24hrs later, for evaluation
(Akt) (Cat. No.9272) and 1:200 rabbit polyclonal anti-p-Akt of:cellviability,ROS levels,NADPHandeNOSexpression,cell
(Ser413) (Cat. No.9271) both purchased from Cell Signaling migration and cell contraction, modulation of miR21 molecular
Technologies (Danvers, MA, USA); 1:200 donkey polyclonal targets (PTEN, PDCD4, Bcl2) and of Akt phosphorylation.
antibody anti-gp91-phox subunit of nicotinamide adenine dinu- Efficacy of miR21 overexpression was verified by RT-PCR, as
cleotide phosphate (NADPH) (Cat. No. sc-5827, Santa Cruz reportedabove,atbothtwoandfourdaysfollowingtransfection.
Biotechnology, Santa Cruz, CA, USA); 1:200 mouse monoclonal Six experiments were performed forthese studies.
anti-endothelialnitricoxidesynthase(eNOS),Cat.No 5880,Cell
Signaling; 1:200 rabbit polyclonal antibody anti-phospho-eNOS 5.IstheNPR-CdrivencAMP/ProteinkinaseA(PKA)/CREB
(Ser1177)Cat.No9571,CellSignaling;1:200rabbitmonoclonal
axis related to miR21 and pAkt regulation in CASMCs
antibody anti-phosphatase and tensin homolog (PTEN) (Cat.
upon CC2238/aANP exposure?
No 9559, Cell Signaling); 1:200 rabbit monoclonal anti-pro-
To explore the role of cAMP/PKA/CREB axis in CC2238/
grammed cell death protein 4 (PDCD4), Cat. No 9535, Cell
aANPdependenteffectsonCASMCviability,weevaluatedboth
Signaling; 1:200 rabbit polyclonal anti-B-cell lymphoma 2 (Bcl2),
miR21andAktphosphorylationexpressioninthefollowingsetsof
Cat. No sc-492, Santa Cruz Biotechnology; 1:5000 anti-b-actin
experiments:
(Cat. No A5441, Sigma) used as housekeeping gene. Following
threewashesof10min.inTBS-T,membraneswereincubatedfor N
-CC2238/aANPwasco-incubatedwith10mMFSK(inorder
1 hr at room temperature with 1:5000 horseradish peroxidase-
to increase intracellular cAMP levels), as previously reported
conjugatedsecondaryantibodygoatanti-rabbit(Cat.Nosc-2004,
[13].Attheendofstimulation,weassessedexpressionlevelsof
Santa Cruz) or goat anti-mouse (Cat. No sc-2005, Santa Cruz)
miR21and,24hrslater,ofAktphosphorylation(asdescribed
diluted in 5% BSA. After three washes of 10min. in TBS-T,
above).
signals were revealed with an enhanced chemiluminescence N
detection system (Luminata Crescendo, Millipore, Darmstadt, -CC2238/aANP was co-incubated with 10mM FSK and
Germany)andtheimmunoreactivityofbandswasvisualizedona 15mMDihydrochloridehydrate(H89,PKAinhibitor,Sigma).
high-performance chemiluminescence apparatus (ChemiDoc MP Modulation of both miR21 expression and pAkt phosphory-
System, Bio-Rad). Protein bands were scanned and quantified lation levels wasassessed asspecified above.
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T2238C/aANPVariantandCoronaryArterySmoothMuscleCells
N
-CC2238/aANPwasco-incubatedwith10mMFSKfollowing expression and a significant decrease of phosphorylated eNOS
either 60 or 120min. pre-incubation with 25 mM p300/ expression (Fig.1D).
CREB(CBP) (CREB inhibitor, Sigma). Modulation of both CC2238/aANPinducedamarkedincreaseofcellmigration,as
miR21 expression and pAkt phosphorylation levels was comparedtoTT2238/aANP(Fig.2A),consistentlywithprevious
assessed asspecified above. observations obtained in ECs [16]. A significant increase of cell
contraction was detected upon CC2238/aANP exposure
(Fig.2B).
Statistical Analysis
Continuous variables are expressed as mean6SEM. Compar- Investigation of the signaling involved in the negative
isonsbetween2groupswereperformedusingStudentttest.When
effects of CC2238 vs TT2238/aANP in CASMCs
theanalysiswasadjustedforthemultiplicityofcomparedgroups,
ReleaseofcAMPlevelswasdramaticallyreducedbyCC2238/
1-way ANOVA followed by Bonferroni post hoc test was
aANP, in contrast to wild type aANP (Fig.3A). Levels of cGMP
performed. SPSS statistical software (SPSS Inc, Chicago, IL,
wereremarkablyincreasedbybothwildtypeandvariantaANPat
version 12.0)was usedforstatistical analysis.
all tested concentrations (Fig. 3B, S2A). Blocking of NPR-A with
A p,0.05 wasconsidered significant.
anantindidnotavoidthedeleteriouseffectsofCC2238/aANPon
cell viability, and it was associated with a marked cAMP levels
Results
decrease (S2B). Degree of Akt phosphorylation was significantly
reduced by aANP variant only, similarly to what has been
EffectsofexposureofTT2238-andCC2238/aANPoncell
previously reported inECs [13] (Fig.3C).
viability, oxidative stress, migration and contraction in
Based on previous findings showing involvement of miR21
CASMCs
downregulation in the anti-proliferative effect of wild type aANP
Cell viability was significantly impaired in cells exposed to in aortic SMCs [3], we compared degree of miR21 modulation
CC2238/aANP (Fig.1A, 1B, S1A). Consistently, apoptosis and under either TT2238- or CC2238/aANP exposure. A marked
necrosis were significantly increased by variant aANP (Fig.1B, suppression of miR21 expression was revealed upon exposure to
S1A). ROS levels were also markedly increased (Fig.1C), along CC2238/aANP,whereasTT2238/aANPreducedmiR21expres-
with a significant increase of gp91-phox subunit of NADPH sion by 50% (Fig.3D). Notably, the TT2238/aANP effect on
Figure 1. Effects of exposure to either TT2238- or CC2238/aANP on viability and oxidative stress in CASMCs. A. Cell vitality as
determinedbytrypanblue(n=4);B.CellvitalityasassessedbyFACS(n=4):representativescatterplotsareshownwithpercentagesoflivecells
(bottomleft),earlyapoptoticcells(upperleft),lateapoptoticcells(upperright),necroticcells(bottomright);C.ROSlevels(n=6);D.NADPHand
eNOSexpressionlevels(n=3).Representativewesternblotsareshown;barsrepresentresultsofdensitometricanalysis.Control(CTR):untreatedcells;
positivecontrol:cellstreatedwithHO .¤p,0.01;forallothercomparisonsp,0.0001.
2 2
doi:10.1371/journal.pone.0113108.g001
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T2238C/aANPVariantandCoronaryArterySmoothMuscleCells
Figure2.EffectsofexposuretoeitherTT2238-orCC2238/aANPoncellmigrationandcellcontraction.Photosofcellmigration(A)and
ofcellcontraction(B)uponexposureofCASMCstoeitherTT2238/aANPorCC2238/aANPascomparedtocontrol(CTR).Resultsareplottedinthe
graphsatthebottomofthefigure.Numberofexperiments=4.*#p,0.0001.
doi:10.1371/journal.pone.0113108.g002
miR21wasmediatedbycGMPdependentkinase(S3),confirming CC2238/aANP in CASMCs, we overexpressed miR21 in cells
previous data [3]. exposed to variant aANP. Overexpression of miR21 was
KnowntargetsofmiR21areBcl2,PTENandPDCD4thatare confirmed by RT-PCR (S6). Concomitant overexpression of
allinvolvedincellapoptosis,proliferationandgrowth(17–19).In miR21 with exposure to CC2238/aANP restored cell viability,
fact, as a consequence of miR21 suppression, CC2238/aANP reducedcellapoptosisandnecrosis(Fig.S1C),reducedROSlevels
reduced Bcl2 expression and significantly increased both PTEN (Fig.5A), rescued phosphorylated Akt levels (Fig.5B), increased
and PDCD4 expression levels (Fig.3E), as key mechanisms eNOS and reduced NADPH expression (Fig.5C, 5D). In this
underpinning itsdeleterious effects. experimental condition, Bcl2 expression increased whereas both
PTEN and PDCD4 expression decreased as expected (Fig.6).
Impact of NPRC gene silencing on detrimental effects Overexpression of miR21 reduced cell migration stimulated by
induced by CC2238/aANP CC2238/aANP,whereasitwasunabletocounteractthedegreeof
cellcontraction dependent on CC2238/aANP (S7).
Silencing of NPR-C gene abolished the effects induced by
CC2238/aANPoncellviability,cellapoptosisandnecrosis(S1B). Of note, miR21 overexpression alone was able to induce the
expected changes on its own targets and on phosphoAkt levels
It also blunted ROS levels accumulation induced by CC2238/
aANP (Fig.4A). Both Akt phosphorylation (Fig.4B) and miR21 (Fig.5B, Fig. 6). Furthermore, the known stimulating effect on
VSMC contraction [3]was confirmed(S7).
expression (Fig.4C) were restored. Consequent changes of
expression of miR21 related targets (PDCD4, Bcl2, PTEN) are
shown in S4 (panels A–C). In addition, NADPH and eNOS ROS levels directly influence CASMC migration and
protein expression were both rescued (S4, panels D, E). Both contraction upon CC2238/aANP exposure
increased migration and increased contraction dependent on As shown in S8, apocynin abolished the stimulation of both
CC2238/aANPwere abolished (S5). migrationandcontractionuponexposuretoCC2238/aANP.The
contribution of increased cAMP levels to migration and contrac-
Impact of miR21 overexpression in the presence of tion (tested in the presence of FSK) was comparable to that of
CC2238/aANP increased oxidative stress.
In order to test whether the dramatic reduction of miR21
expression levels is involved inthe detrimental effects induced by
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T2238C/aANPVariantandCoronaryArterySmoothMuscleCells
Figure3.InvestigationofthesignalinginvolvedinthenegativeeffectsofCC2238/aANPinCASMCs.cAMP(A)andcGMP(B)levels
(number of experiments=12) (for all comparisons p,0.0001); C. pAkt/Akt levels with representative western blot and results of densitometric
analysis(n=4);D.hsa-miR21expressionlevelsasdeterminedbyRT-PCR(n=6)(forallcomparisonsp,0.0001);E.miR21moleculartargetsexpression
levelsundereitherTT2238/aANPorCC22338/aANPexposure(n=3).Representativewesternblotsareshown;barsrepresentresultsofdensitometric
analysis.(¤p,0.01;forallothercomparisonsp,0.0001).CTR=control.
doi:10.1371/journal.pone.0113108.g003
cAMP/PKA/CREB axis modulates miR21 and phosphoAkt miR21 suppression in mediating the deleterious effects on
expression levels upon CC2238/aANP in CASMCs CASMC viability exerted by CC2238/aANP; 2) first demonstra-
Co-incubation of CC2238/aANP with FSK allowed a signif- tion that NPR-C/cAMP/PKA/CREB axis is involved in modu-
lationofbothmiR21andAktphosphorylationinCASMCsupon
icantincreaseofbothmiR21andphosphoAktexpressionlevels,as
comparedtocellstreatedwithCC2238/aANPonly(Fig.7A,7B, CC2238/aANP; 3) first evidence that CC2238/aANP is a
7C).InthepresenceofconcomitantexposuretoCC2238/aANP, significant promoter ofboth CASMCmigration andcontraction,
mainly dependent on ROS accumulation, and therefore it exerts
FSKandH89bothmiR21andphosphoAktlevelswereunableto
vascular functional properties different from those of regular
increaseasmuchasinthepresenceofFSKonly(Fig.7).Thesame
resultswereobtainedwhencellswereexposedtoCC2238/aANP, aANP (S9).
ANP is involved in the control of cardiac and vascular
FSK and CREB inhibitor (Fig.7). These data demonstrate that
remodeling by performing anti-proliferative effects on cardiac,
inhibitionofcAMP/PKA/CREBaxisdoesnotallowtherecovery
endothelialandSMcells[1,2,20].Functionalderangementsdueto
of both miR21 and phosphoAkt expression levels in thepresence
of CC2238/aANP. either circulating peptide levels or peptide structure lead to
pathological stateswithinthecardiovascular system [21].
The common T2238C/aANP molecular variant has recently
Discussion
emergedasanovelcardiovascularriskfactorforitsabilitytoalter
The present study explored the impact of CC2238/aANP in the physiological beneficial properties of regular aANP. Apart
VSMCs,afundamentalcellularelementforvesselwallphysiology fromthepreviouslyidentifiedeffectsonECviabilityandfunction
anddisease, andit demonstrated forthefirst timethat CC2238/ [13,16], wenowdemonstrate itsdeleterious actions inVSMCs.
aANPisahighlydeleteriousfactorforcoronaryarterySMCs,as The effects of CC2238/aANP in CASMCs were mediated by
compared towild typeaANP. NPR-C activation, similarly to what has been first discovered in
The main original findings of the current set of data can be ECs, and strictly dependent on the higher affinity binding of
summarizedasfollows:1)identificationofastrongcontributionof CC2238/aANPwithNPR-C[13].Infact,CC2238/aANPledto
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T2238C/aANPVariantandCoronaryArterySmoothMuscleCells
Figure4.ImpactofNPR-CgenesilencingondetrimentaleffectsinducedbyCC2238/aANPinCASMCs.A.ROSlevels(n=6);B.pAkt/Akt
levelswithrepresentativewesternblotandresultsofdensitometricanalysis(n=3);C.miR21expressionlevelsinNPR-Cgenesilencedcellsupon
exposuretoCC22338/aANP(n=6).Control(CTR):untreatedcells;positivecontrol:cellstreatedwithH O .siRNA1andsiRNA2=smallinterfering
2 2
RNAsforNPR-Cgenesilencing.Forallcomparisonsp,0.0001.
doi:10.1371/journal.pone.0113108.g004
a significant reduction of CASMC viability and of cAMP levels, aortic SMCs, whereas overexpression of miR21 restored VSMC
dependent on NPR-C driven adenylate cyclase inhibition, even proliferation [3]. In our experimental setting, we observed that
when NPR-A was blocked by the use of anantin. Consistently, miR21 levels were completely suppressed by CC2238/aANP as
NPR-C gene silencing abolished all effects due to aANP variant. compared to wild type aANP (that caused 50% reduction).
Ofnote,thefunctionalconsequencesofpathologicalactivationof Accordingly, expression levels of miR21 molecular targets were
NPR-C by CC2238/aANP strongly differ from those obtained remarkably changed by aANP variant. The miR-21 suppression
through physiological stimulation with its agonist, i.e. CNP, appearedtobetightlyrelatedtoallnegativeeffectsobservedupon
mainly consisting inbeneficial effects on vascularcells [22]. CC2238/aANP. In fact, overexpression of miR21 rescued
The microRNA-21, that is strongly involved in the effects expression levels of its molecular targets and of Akt phosphory-
dependent on CC2238/aANP in CASMCs, is emerging as a lation, while restoring cell viability. Our results imply that
fundamental protective factor for maintaining vitality and suppression of miR21 upon CC2238/aANP contributes to cause
proliferation in several cell types and it has been found to be detrimental effects in VSMCs, whereas reduction of miR21,
highly expressed in all main types of cardiovascular cells [23]. It mediatedbycGMPdependentkinase,producesthephysiological
plays important roles in VSMC proliferation and apoptosis anti-proliferative effectof wildtype aANP.
through the targeting of PTEN, Bcl2 and PDCD4 proteins [17– Notably, we support evidence that miR21 belongs to Akt
19]. The known anti-proliferative effect of wild type aANP has regulatory network incardiovascularcells, asan upstream factor,
been previously shown to associate with a decrease of miR21 in duetoitsabilitytoinhibitPTENandtoexert,throughconsequent
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Figure 5. Impact of miR21 overexpression in CASMCs in the presenceof CC2238/aANP.A. ROS levels (n=6); B. pAkt/Akt levels with
representativewesternblotandresultsofdensitometricanalysis(n=3);eNOS(C)andNADPH(D)expressionlevelswithrepresentativewesternblot
andresultsofdensitometricanalysisincellsoverexpressingmiR21andconcomitantlyexposedtoCC22338/aANP(n=3).Control(CTR):untreated
cells;positivecontrol:cellstreatedwithH O .Forallcomparisonsp,0.0001.
2 2
doi:10.1371/journal.pone.0113108.g005
increased Akt activity, important effects on cell proliferation provide original evidence that deregulated activation of NPR-C,
[17,24]. Infact, bothPTENandAktrepresent keymoleculesfor upon exposure to variant aANP, suppresses expression levels of
cell growth and survival, as well as for development of many miR21 in CASMCs. In fact, cells exposed to CC2238/aANP, in
cardiovascular diseases [25–27]. In our experimental context, theabsenceofNPR-C,showedregularexpressionlevelsofmiR21.
miR21 suppression, dependent on CC2238/aANP, led toPTEN Previous studies have shown the ability of cAMP/PKA to
upregulation.Inturn,miR21overexpressionreducedPTENand, regulate expression levels of few miRNAs, such as miR1 [28],
in the presence of CC2238/aANP, prevented the increase of miR335 [29], and miR375 [30]. Herein, we report the first
PTENexpression.Finally,Aktphosphorylationwasmodulatedin evidence that cAMP/PKA/CREB pathway modulates miR21
a manner consistentwith PTENchanges. expression in CASMCs. In fact, PKA inhibition by H89 did not
CurrentknowledgeonreceptorsinvolvedinmiRNAexpression allow therecovery ofmiR21levels inthepresenceof FSK,upon
modulationisscarce.ActivationofNPR-Ahasbeensuggestedas stimulation with CC2238/aANP. Inhibition of CREB, cAMP
the responsible mechanism for modulation of several miRNAs, responsiveelement, byp300/CREB(CBP) ledtoidentical results.
including miR21, by wild type aANP in aortic SMCs [3]. We
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Figure6.miR21targetsexpressionlevelsundermiR21overexpressionandconcomitantexposureofCASMCstoCC2238/aANP.
Representativewesternblotsareshown;barsrepresentresultsofdensitometricanalysis.Numberofexperiments=4.Forallcomparisonsp,0.0001.
CTR=control.
doi:10.1371/journal.pone.0113108.g006
Thus,wesupportknowledgeontheabilityofcAMP/PKA/CREB important achievement of the current study. An increased
axis toregulate expression of miRNAs invascularcells. vasoconstriction, along with the previously reported endothelial-
Migration and contraction are fundamental processes for dependent impaired vasorelaxation [13], constitutes a rational
vascular remodeling, proliferation and disease [31,32]. Herein, pathologicalsubstrateforhigherpredispositiontoplaqueinstabil-
we show that CC2238/aANP is a strong promoter of CASMC ityandforanaugmentedrate ofacutecoronaryandcerebrovas-
migration and contraction, mainly dependent on ROS accumu- culareventsobservedinCC2238/aANPcarriers,asdocumented
lation. In fact, decreases ofROS levels prevented bothmigration by several invivo studies [7–12]. The recent demonstration that
and contraction in the presence of aANP variant. The strong VSMCs stabilize atherosclerotic plaques [6] and that VSMC
contributionofROStovascularcellmigrationandcontractionare death and reduced proliferation promote plaque instability [36]
known [33,34]. further support our conclusions on the potential mechanisms,
ItisalsoworthwhileobservingthatmiR21,whenoverexpressed involving VSMCs, underpinning increased rate of acute cardio-
in the presence of aANP variant, antagonized the effect of the vascular eventsinCC2238/aANPcarriers.
latterwithregardtomigrationbutitwasunabletoantagonizethe Fewlimitationsofthestudyneedtobeacknowledged.Wewere
contractilepropertyofCC2238/aANP.Thisevidenceisconsistent unabletoreproduceinvitro,forobvioustechnicallimitations,the
withthenotionthatmiR21exertsVSMCcontractionthroughan effects of the CT heterozygous peptide. On the other hand, it is
independent activation ofDOCKproteins [35]. assumed that heterozygous subjects carry 50% of both aANP
Notably, CC2238/aANP exerted CASMC contraction despite forms.
miR21suppression.Thedemonstrationofasignificantlyincreased Inaddition,althoughthecurrentin vitrodatawereobtainedin
vasoconstrictiondependentonCC2238/aANP,incontrasttothe
acelllinerelevantforthehumandisease,wecannotextendthem
known vasodilation promoted by wild type aANP, represents an
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T2238C/aANPVariantandCoronaryArterySmoothMuscleCells
Figure7.EvidencethatcAMP/PKA/CREBaxisregulatesmiR21expressionandAktphosphorylationinCASMCs.A.miR21expression
levelsuponconcomitantexposureofCASMCsto:CC2238/aANPandFSK;CC2238/aANP,FSKandH89;CC2238/aANP,FSKandCREBinhibitorp300/
CREB(CBP);thelatterwaspre-incubatedeither60or120min.followedbyexposuretoCC2238/aANPandFSK;BandC.pAkt/Aktexpressionlevelsin
thesameexperimentalconditions.Numberofexperiments=6.Forallcomparisonsp,0.0001.
doi:10.1371/journal.pone.0113108.g007
to other vascular beds. Finally, the invivo implications of our For all comparisons of CC2238/aANP vs other treatments: p,
findings remain tobeassessed. 0.0001.
In conclusions, based on the current results, CC2238/aANP (TIF)
exertsdeleteriouseffectsonviabilityandfunctionofCASMCsand
Figure S2 A. cGMP levels in response to different
it does it through binding with NPR-C, involvement of cAMP/
concentrations of both TT2238- and CC2238/aANP
PKA/CREB/miR21 pathway and marked increase of oxidative
(number of experiments=5); B. Left panel=CASMC
stress. The reduced proliferation and increased contraction of
vitality as assessed by trypan blue upon CC2238/aANP
CASMCs maycontributetoexplaintheaugmentedrateofacute
exposure in the presence of anantin; levels of cGMP
coronary events in CC2238/aANP carriers. Pharmacological
(middlepanel)andofcAMP(rightpanel)uponCC2238/
targetingofmolecularpathwaysactivatedbyCC2238/aANPmay
aANP exposure in the presence of anantin (number of
beproposedasavaluablestrategytoreducecardiovascularriskin experiments=4);For allcomparisons: p,0.05.
subjects bearingthisaANPvariant.
(TIF)
Supporting Information FigureS3 miR21 expression levels upon TT2238/aANP
eitherintheabsenceorinthepresenceofcGKinhibitor.
FigureS1 GraphicalrepresentationofFACSanalysisin Number ofexperiments=3.For all comparisons: p,0.05.
thedifferentexperimentalsettings.Barsrepresentcountsof (TIF)
live cells, early and late apoptotic cells, necrotic cells upon
Figure S4 Impact of NPR-C gene silencing on protein
exposure of CASMCs to CC2238/aANP alone (A), in the
expression levels upon concomitant exposure to
presence of NPR-C gene silencing (B), and in the presence of
CC2238/aANP. miR21 targets expression levels (A, B, C);
miR21 overexpression (C). siRNA1 and siRNA2=small interfer-
NADPH (D) and eNOS (E) expression levels. Representative
ingRNAsforNPR-Cgenesilencing.Numberofexperiments=4.
western blots are shown; bars represent results of densitometric
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