Barquín, Magda Heras, M. Teresa Pérez-García and José R. López-López Pilar Cidad, Alejandro

32 Pages·2010·2.05 MB·English

Characterization of Ion Channels Involved in the Proliferative Response of
Femoral Artery Smooth Muscle Cells
Pilar Cidad, Alejandro Moreno-Domínguez, Laura Novensá, Mercé Roqué, Leire
Barquín, Magda Heras, M. Teresa Pérez-García and José R. López-López
Arterioscler Thromb Vasc Biol 2010, 30:1203-1211: originally published online
March 18, 2010
doi: 10.1161/ATVBAHA.110.205187
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Cell Biology/Signaling
Characterization of Ion Channels Involved in the
Proliferative Response of Femoral Artery Smooth
Muscle Cells
Pilar Cidad, Alejandro Moreno-Domínguez, Laura Novensa´, Merce´ Roque´, Leire Barquín,
Magda Heras, M. Teresa Pe´rez-García, Jose´ R. Lo´pez-Lo´pez
Objective—Vascularsmoothmusclecells(VSMCs)contributesignificantlytoocclusivevasculardiseasesbyvirtueoftheir
abilitytoswitchtoanoncontractile,migratory,andproliferatingphenotype.Althoughtheparticipationofionchannels
in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined
becauseoftheirlargemoleculardiversity.Weobtainedaglobalportraitofionchannelexpressionincontractileversus
proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant
changes that we observed.
Methods and Results—High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed
in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs
obtainedfromexplants.mRNAexpressionchangesshowedagoodcorrelationbetweenthe2proliferativemodels,
with only 2 genes, Kv1.3 and Kv(cid:1)2, increasing their expression on proliferation. The functional characterization
demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits
migration and proliferation.
Conclusion—These findings establish the involvement of Kv1.3 channels in the PM of VSMCs, providing a new
therapeutical target for the treatment of intimal hyperplasia. (Arterioscler Thromb Vasc Biol. 2010;30:1203-1211.)
Key Words: gene expression (cid:1) ion channels (cid:1) restenosis (cid:1) vascular biology (cid:1) vascular muscle (cid:1) Kv1.3 channels
(cid:1) vascular remodeling
Vascular smooth muscle cells (VSMCs) are differenti- programs to readjust cellular activity to mechanical and
atedcellsthatregulatevesseldiameteranddetermine hormonal factors.1–3
tissue perfusion. However, they can exhibit a variety of
See accompanying article on page 1073
functionally dissimilar phenotypes. In response to local
The switch in ion transport mechanisms associated with
cues, VSMCs experience a phenotypic modulation (PM),
PM is getting increasing amounts of attention. Coordinate
with profound and reversible changes leading to prolifer-
changes in ion channels are an integral component of
ation, migration, and secretion of extracellular matrix
VSMCplasticity,astheycanredirectbiochemicalactivity
components.1 This plasticity is essential for injury repair,
toward new functional responses.4,5 Moreover, both con-
but it also contributes to the development and progression
tractileandproliferativesignalsrequirespecificchangesin
ofvasculardiseaseinresponsetoabnormalenvironmental intracellular [Ca2(cid:1)] and membrane potential that are de-
signals. It is becoming evident that contractile and prolif-
termined by the ion channels expressed in VSMCs. Re-
erative phenotypes represent extreme cases of a spectrum
modeling of several ion channels has shown to be func-
of phenotypes that may coexist as the result of a develop- tionally important for the PM of VSMCs in several
mentally regulated genetic program constantly modulated preparations.5–10 These data contribute to our understand-
by environmental cues. This explains both a relatively ing of VSMC modulation, but also, importantly, they can
stable expression of certain transcriptional programs in providenewtargetsforthetreatmentofvasculardisorders.
different VSMCs and a marked plasticity of these cells, Todate,studiesofionchanneldistributionwithinspecific
including the ability to respond with different genetic vascular beds and the modifications of ion channels on
Receivedon:April3,2009;finalversionacceptedon:March10,2010.
FromDepartamentodeBioquímicayBiologíaMolecularyFisiologíaeInstitutodeBiologíayGene´ticaMolecular,UniversidaddeValladolid
yConsejosuperiordeInvestigacionesCientificas,Valladolid,Spain(P.C.,A.M.-D.,M.T.P.-G.,J.R.L.-L.);ServiciodeCardiología.InstitutClinic
delTo`rax,HospitalClinic,InstitutodeInvestigacionesBiomedicalsAugustPiiSunyer,UniversidaddeBarcelona,Barcelona,Spain(L.N.,M.R.,
L.B.,M.H.).
CorrespondencetoJose´ Ramo´nLo´pez-Lo´pez,DepartamentodeBioquímicayBiologíaMolecular,UniversidaddeValladolid,EdificioInstitutode
BiologíayGene´ticaMolecular,c/SanzyFore´ss/n,47003Valladolid,[email protected]
©2010AmericanHeartAssociation,Inc.
ArteriosclerThrombVascBiolisavailableathttp://atvb.ahajournals.org DOI:10.1161/ATVBAHA.110.205187
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1204 Arterioscler Thromb Vasc Biol June 2010
remodeling have been limited to a small number of tionsofthepatch-clamptechniquewereusedforfunctionalstudies
candidate subunits believed to be of importance. In addi- inisolatedVSMCs8,12
tion,thecomplexityoftheircharacterizationintheinvivo
models has led most investigators to extrapolate from the Migration and Proliferation Studies
data obtained from cultured VSMCs as a proliferative Migration of cultured VSMCs was studied by a scratch assay and
phenotype, because VSMCs in intimal hyperplasic lesions proliferation was determined by 5-bromo-2(cid:3)-deoxyuridine (BrdU)
resemble dedifferentiated myofibroblasts or cultured incorporation.
VSMCs. However, the emerging picture regarding the
phenotypic regulation of VSMCs shows a large degree of Results
diversity, reflecting both intrinsic variability of VSMCs
Expression Profile of Ion Channel Genes in
among vascular beds and the specific responses to the
Femoral VSMCs
different proliferative signals present in cultured cells and
The transcriptional expression levels of 87 ion channel
in the in vivo lesions.
subunits in VSMCs from mouse femoral arteries were
Here, we used a quantitative approach to obtain a global
investigatedbyreal-timepolymerasechainreaction.These
portraitofionchannelgeneexpressionincontractileVSMCs
channelgenesinclude(cid:2),(cid:1),and(cid:3)subunitsofK(cid:1)channels
from mouse femoral arteries and their changes on PM in 2
and (cid:2)subunits of voltage-dependent Ca2(cid:1) channels, Cl(cid:4)
proliferative models: an in vivo model of neointimal
channels, and Trp channels. The expression levels of
hyperplasia induced by endoluminal lesion11 and an in
markers of VSMCs (calponin, Cnn1) and endothelium
vitro model using cultured VSMCs from arterial explants.
Two genes (Kv1.3 and Kv(cid:1)2) showed a concordant up- (endothelial nitric oxide synthase, Nos3, and von Wille-
brand factor), were also explored, as well as other endog-
regulationinbothmodels,andtheexpressionandfunction
enous controls (Gapdh, B2m, Hprt-1, and Klf5). Expres-
ofKv1.3proteinswasexplored.Electrophysiologicalstud-
sion of 26 channel genes was undetectable after 40 cycles
ies in cultured VSMCs and VSMCs from injured arteries
of amplification under all conditions.
showanincreasedfunctionalexpressionofKv1.3currents.
Therelativeabundanceofthe54channelgenesfoundin
Pharmacological or genetic Kv1.3 blockade inhibits cul-
the control preparation (mRNA from intact femoral arter-
tured VSMC migration and proliferation, and this inhibi-
ies, with endothelium, C ) is shown in Figure 1. Genes
tion cannot be mimicked by VSMC depolarization. Fi- E(cid:1)
are grouped by families, and their expression levels are
nally, the analysis of other vascular beds confirms that
upregulationofKv1.3associateswithPM,suggestingthat normalized to the endogenous control ribosomal protein
they can represent a good therapeutical target by which to 18S.Wedetectedexpressionof1Cl(cid:4)channel(Clcn3)and
control VSMC proliferation. several inward rectifier and 2-pore domain K(cid:1) channels.
We found expression of all members of the Ca2(cid:1)-
dependentK(cid:1)channels(K )family,withtheexceptionof
Ca
Materials and Methods themaxiK(BK )(cid:1)subunitBK(cid:1)2;ofthem,theBK(cid:2)and
Ca
AnexpandedMaterialsandMethodssectionisgiveninthesupple- BK(cid:1)1subunitswereamongthemostabundantlyexpressed
mentalmaterial,availableonlineathttp://atvb.ahajournals.org. channel genes. Within the voltage-dependent K(cid:1) channels
(Kv channels), we found expression of several accessory
Animals subunits, as well as pore-forming (cid:2)subunits of members
Bloodpressurenormal(BPN)mice(TheJacksonLaboratory)were of the Kv1 to Kv4 and Kv11 subfamilies. Voltage-
maintainedwithinbredcrossingintheanimalfacilitiesoftheschools dependent Ca2(cid:1) channels are represented by Cav1 and
ofmedicineoftheuniversitiesofValladolidandBarcelona.Unilat-
Cav3 subfamily members. Finally, we detected expression
eral transluminal injury of femoral arteries was performed by
passageofa0.25-mm-diameterangioplastyguidewire,aspreviously ofalargenumberofTrpchannelsoftheTrpc,Trpv,Trpm,
described.11 andTrppsubfamilies,withTrpp2beingthemostabundant
transcript.
VSMC Isolation Changes in the expression profile of ion channels
Micewerekilledbydecapitationafterisofluoraneanesthesia.Fem- induced by the switch to a proliferative phenotype were
oralarteriesweredissectedandcleanedofconnectiveandendothe- studiedinVSMCsobtainedfromexplantsofendothelium-
lialtissues,andtheywerestoredforRNAextractionoruseddirectly denuded femoral arteries and kept in culture (in vitro
toobtainfreshdispersedVSMCsorculturedVSMCsaspreviously
model) and in endoluminal lesion-induced intimal hyper-
described.8,12
plasia (in vivo model), in which expression changes were
studied at 3 different times after lesion (48 hours, 1 week,
RNA Expression Profile
and 4 weeks). Each model had its own control: endo-
TaqMan low-density arrays (Applied Biosystems) were used to
thelium-free arteries (C ) for the in vitro model and
simultaneously quantify the expression of 96 genes per sample.12 E(cid:4)
Each sample derived from (cid:2)500 ng of mRNA from contractile or endothelium intact arteries (CE(cid:1)) for the in vivo model.
proliferatingVSMCs. Differencesbetweenthese2controlsaresummarizedintheinset
inFigure1,wheregeneswhoseexpressionwasmodifiedinthe
Protein Expression and Function CE(cid:4)samples(relativetoCE(cid:1))areshown.
Selective antibodies were used for protein detection with immuno- Changes in proliferation were analyzed by 2 methods.
logic methods.8,11 The whole-cell and perforated-patch configura- First, we studied, for each individual gene, the differences
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Cidad et al Ion Channels and VSMC Proliferation 1205
Figure1.Relativeabundanceoftheionchannel
genesstudiedincompletefemoralarteries(C )
E(cid:1)
expressedas2(cid:4)(cid:5)Ct,where(cid:5)Ct(cid:6)Ct (cid:4)Ct .
channel 18s
Eachbaristhemean(cid:7)SEMof10determinations
obtainedin5duplicateassays.Genesaregrouped
byfamilies(seeonlinesupplementaldataforthe
listofchannelgenes).Theinsetshowsfold
changesinexpressioninendothelium-freearteries
(C )expressedaslog(2(cid:4)(cid:5)(cid:5)Ct),where
E(cid:4)
(cid:5)(cid:5)Ct(cid:6)(cid:5)Ct(C )(cid:4)(cid:5)Ct(C ).Inallfigures,*P(cid:8)0.05,
E(cid:4) E(cid:1)
**P(cid:8)0.01,and***P(cid:8)0.001.
betweencontrolandproliferationinthe2modelsbyusing sificationsinthemodelofendoluminallesion(Figure2A).
the 2(cid:4)(cid:5)(cid:5)Ct relative quantification method13 (Supplemental Groups of genes with similarity in the pattern of expres-
Figure III). Second, a 2-way hierarchical clustering anal- sion (indicated by the correlation coefficient) are high-
ysis of genes and different experimental conditions14 was lighted in Figure 2. Within downregulated genes, we
performed and visualized with the Treeview software identified clusters of genes with late decrease expression
(Figure 2). This analysis categorized genes according to (Figure 2, box A), early decrease followed by a partial
theirresponsestoPMintoupregulatedanddownregulated recovery (box B), or time-independent decrease (box C).
genes and also demonstrated time-course–related subclas- Similarly, PM-upregulated genes can show a late increase
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1206 Arterioscler Thromb Vasc Biol June 2010
Figure2.A,Two-wayhierarchicalagglomerativeclusteringappliedto59genes(horizontally)andto5contractileVSMCssamples
(C1toC5),2samplesat48hoursafterendoluminallesion,2samplesafter1week,and3samplesafter4weeks(vertically).The
inputdatawerethe(cid:5)Ctvaluesforthegenes.Colorpatchrepresentstheexpressionlevelforeachgeneandsample.Expression
levelswerenormalizedwithineachsample,withascalerangingfrombrightgreen(lowest)tobrightred(highest).Missingvalues
areshownasgraypatches.Thelengthofthetreebranchesisproportionaltothecorrelationofthegeneexpressionpattern,and
someoftheseclustersandcorrelationcoefficientsareindicated.B,SamerepresentationasinA,butcomparingthecontractile
VSMCs(labeledhereastissuesT1toT3)withtheculturedVSMCs(C1toC3).C,Diagramshowinggenesexhibitingsignificant
expressionchanges(upregulation,redfontordownregulation,greenfont)inendoluminallesionsamplesandinculturedVSMCs,
whenanalyzedwiththeT-REXsetoftoolsoftheGEPASsuite(seeSupplementalMethods).Thelightgrayintersectioncontains
thegenesshowingthesamesignificantchangesinbothmodels.
(boxD),anearlyincreasewithpartialrecovery(boxE),or obtained a significant variation in the expression of 22
a sustained increase (box F). Following these criteria, the genes in the in vivo model and 29 genes in the in vitro
hierarchical clustering of the genes whose expression was model (Figure 2C). The endoluminal lesion induced a
modified in cultured VSMC (Figure 2B) also identified 2 significant increase in 3 genes (ClCa1, Kv1.3, and Kv(cid:1)2),
groups of genes with decreased (boxes A through C) or andasignificantdecreasein19genes.InculturedVSMCs,
increased (boxes D through F) expression with 7 genes were overexpressed and 22 exhibited significant
proliferation. decrease. The overlapping region in Figure 2C shows the
To discern whether these expression patterns were genes with significant changes in their expression profile
simply arbitrary structures or rather reflect biologically commontobothproliferativemodels(18genes).Ofthem,
significantassociations,weperformedastatisticalanalysis wefocusedonthosewithincreasedexpression(Kv1.3and
of the data with the GEPAS suite (http://gepas.bioinfo. Kv(cid:1)2), as they are more amenable to represent therapeu-
cipf.es/), with false discovery rate (FDR) correction.15We tical targets. We studied the presence, distribution, and
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Cidad et al Ion Channels and VSMC Proliferation 1207
Figure3.A,FamilyofcurrenttracesobtainedinfreshlydissociatedandculturedVSMCswiththeindicatedpulseprotocol.Cur-
rentdensityofthetotaloutwardK(cid:1)currentwascalculatedindepolarizingpulsesto(cid:1)40mV.Kvcurrentdensitywasobtained
afterBK blockwith500nmol/Lpaxilline.Mean(cid:7)SEMof14to18cells.B,TheupperplotshowsthefractionofthetotalKvcur-
Ca
rentrepresentedbyKv1.xcurrents(correolide-sensitive)andKv1.3currents(margatoxin-orPAP1-sensitive)incontractile(tissue)
versusculturedVSMCs.Theamplitudeofthedepolarizationinducedbytheseblockersinperforated-patchrecordingsisrepre-
sentedinthelowerplot.Mean(cid:7)SEMof11to14cells.C,PharmacologicalcharacterizationofoutwardK(cid:1)currentsincontractile
andculturedVSMCs.Shownarecurrent/voltagerelationshipsobtainedin2cellsincontrolconditionsandaftersequentialappli-
cationofpaxilline(500nmol/L),margatoxin(10nmol/L),correolide(5(cid:4)mol/L),andTEA(20mmol/L).TheKv1.3componentisrep-
resentedbytheshadedareas.Thetracesshowcurrentat(cid:1)80mVinbothcellswitheachblocker,andpiechartsrepresentthe
proportionofKv1-Kv4currentsinbothpreparations.
functional contribution of Kv1.3 in both proliferative larger in cultured VSMCs (from 44.12(cid:7)1.6% in tissue to
models. 68.69(cid:7)6.5% in culture, Figure 3B). The increased func-
tional contribution of Kv1.3 channels can account for this
Functional Expression of Kv Channels in change, as revealed by selective Kv1.3 blockers (marga-
Femoral VSMCs toxin and PAP-117,18). Current sensitive to 10 nmol/L
Outward K(cid:1) currents were studied with the whole-cell margatoxinrepresented58.15(cid:7)4.54%oftheKvcurrentin
patch clamp technique in freshly dissociated VSMCs and culturedVSMCsversus10.66(cid:7)2.03%infreshlydispersed
in cultured VSMCs from femoral arteries. Figure 3A VSMCs. Similar results were obtained with 10 nmol/L
shows representative traces obtained in each condition. PAP-1.Thefunctionalcontributionofthesechannelstoset
Both total outward K(cid:1) current density, and the Kv com- restingV wasexploredwiththesameblockers,providing
M
ponent of this current (ie, the 500 nmol/L paxilline-resis- parallel results (Figure 3B, bottom). Figure 3C depicts
tant current16) was significantly larger at all voltages in examplesofthepharmacologicaldissectionoftheoutward
VSMCs in the contractile phenotype, but there were no K(cid:1) currents. In the presence of paxilline (500 nmol/L),
significant differences in the proportion of the two com- application of margatoxin or PAP-1 (10 nmol/L each)
ponents. However, the Kv1 fraction of the current (the allowed quantification of the Kv1.3 component. After
5 (cid:4)mol/L correolide-sensitive current) was significantly Kv1.3 blockade, correolide (5 (cid:4)mol/L) was used to selec-
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1208 Arterioscler Thromb Vasc Biol June 2010
Figure5.A,Representativeexampleofthechangesinpeak
currentamplitudeat(cid:1)40mVinaVSMCisolatedfroman
injuredarterywiththeindicateddrugs.B,Labelingwithanti-
SM22(green)demonstratesthevascularsmoothmusclelin-
eageofthesecells.C,Absolutemagnitudeofthe
margatoxin-sensitivecomponent(I )incontrol(contractile)
Kv1.3
andinculturedandinjuredVSMCs.Mean(cid:7)SEMof11to15
cells.
In addition to the increased proportion of Kv1 currents,
cultured VSMCs also showed a significant decrease in the
Kv2(from36.52(cid:7)2.97%to21.34(cid:7)3.71%)andKv3(from
16.4(cid:7)2.74% to 5.56(cid:7)0.97%) components. The Kv3 com-
ponent was estimated as the fraction of the currents
Figure4.A,ImmunocytochemicalidentificationofKv1.3and blockedby100(cid:4)mol/LTEAaftercorreolide,andtheKv2
Kv1.5proteininfreshlydispersedandculturedfemoral
component was calculated as the difference between
VSMCs.Anti-Kv1.3oranti-Kv1.5labeling(green)iscombined
with4(cid:3),6-diamidino-2-phenylindolestaining(blue)B,Paraffin 20 mmol/L and 100 (cid:4)mol/L TEA-sensitive currents.
sectionsofcontrolandinjuredfemoralarterieswerelabeled
withtheindicatedantibodiesandcounterstainedwithhema-
Kv1.3 Protein Expression in Femoral VSMCs
toxylin.Arrowsindicateinternalelasticlamina(IEL).C,Sum-
marydatarepresentthepercentageoflabeledareaoverthe Changes in Kv1.3 protein expression were explored by
totalvesselarea.Mean(cid:7)SEMof10to25sectionsfrom6to immunocytochemistry in freshly dispersed and cultured
13animals.
VSMCs and by immunohistochemical labeling in sections
of control and injured femoral arteries. We have also
tively block the remaining Kv1 currents. Subsequent studied Kv1.5 protein expression as one example of
application of tetraethylammonium chloride (TEA) channeldownregulatedonPM.Kv1.3labelingincreasedin
(20 mmol/L) identified the amplitude of the Kv2(cid:1)Kv3 cultured VSMCs when compared with freshly dispersed
component. The minimal residual current, which was cells, whereas Kv1.5 labeling disappeared (Figure 4A).
insensitive to correolide and TEA, could reflect Kv4 Figure 4B shows similar results in sections of femoral
current component. The molecular composition of Kv arteries: Kv1.3 staining was weak in control sections, and
currents in each preparation is illustrated in the pie charts. increased after lesion (mainly in the intimal layer),
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Cidad et al Ion Channels and VSMC Proliferation 1209
Figure6.A,EffectofKv1.3blockersonfemoralVSMCmigrationcalculatedasthepercentageofinvadedareainscratchassays.
Imagesshowarepresentativeexperimentwith10nmol/LPAP-1.B,Dose-responseeffectofPAP-1ontheproliferationrateof
culturedfemoralVSMCsmeasuredwithaBrdUincorporationassay.n(cid:6)3to8experimentsineachgroup;SFindicatesserum-
freemedium.C,TwodifferentKv1.3shortinterferingRNAs(siRNAs)wereabletoreducemRNAexpression(normalizedtoGapdh)
andBrdUincorporationintransfectedVSMCscomparedwithmock-transfectedorsiRNAC–transfectedVSMCs(n(cid:6)4).D,
EffectsofKchannelblockersorhigh[K(cid:1)] onrestingV (current-clampexperiments)andonproliferationinhibition(BrdUrdincor-
e M
poration).Mean(cid:7)SEMof8to20determinations.TheinsetshowstheantiproliferativeeffectofMgTx(10nmol/L)incontrol
mediumandin20mmol/LK(cid:1)-containingmedium.n(cid:6)7experiments.
whereaslabelingwithanti-Kv1.5antibodywasdetectedin liferation rate, determined by the number of BrdU(cid:1) cells,
the muscular layer of almost half of the cells in control was significantly decreased by Kv1.3 channels blockade
arteries, decreasing on injury. Functional characterization (Figure 6B through 6D). The effect of PAP-1 inhibiting
of Kv1.3 currents from VSMCs obtained from injured proliferation was dose dependent (Figure 6B) and could
arteries (Figure 5A) showed an augmented expression of be mimicked by selective knockdown of Kv1.3 currents
the Kv1 component (85.2(cid:7)4.5% of the Kv current) due to with short interfering RNA (Figure 6C). PAP-1 and mar-
an increase of the MgTx-sensitive component gatoxindepolarizedVSMCs,butothermaneuversthatalso
(61.5(cid:7)5.2%), very similar to the case with cultured depolarized cultured VSMCs, such as increasing [K(cid:1)] or
e
VSMCs (see Figure 3B). Moreover, increased Kv1.3 blockade of Kv2 or BK channels, did not affect prolif-
Ca
current was not simply reflecting downregulation of other eration (Figure 6D). Moreover, proliferation inhibition by
Kv channels, as Kv1.3 current density increased in both 10 nmol/L margatoxin was attenuated but still significant
proliferation models (Figure 5C). in the presence of 20 mmol/L K(cid:1)e (Figure 6D, inset).
Effects of Kv1.3 Blockade on VSMC Migration
Discussion
and Proliferation
The upregulated functional expression of Kv1.3 could This work provides a comprehensive study of the expres-
reflect a link between the channel and the establishment sion pattern of ion channels in VSMCs, using a quantita-
and maintenance of the proliferative phenotype. To ex- tive high-throughput technique, with the goal of elucidat-
plorethis,westudiedtheeffectofKv1.3currentblockade ing PM-associated changes. We defined the relative
on the ability of cultured VSMCs to migrate and prolifer- expression of the ion channel genes studied and compared
ate. Migration was determined in confluent femoral this profile among different vascular beds, observing a
VSMCs by a scratch migration assay. After 24 hours in continuous pattern that correlates with the size of the
serum-free medium alone (control) or with 10 nmol/L arteries (see Supplemental Figure IV), in agreement with
PAP-1 or 10 nmol/L margatoxin, the invaded area was previousdatafocusedonindividualchannels.19–23Charac-
significantly larger in control cells than in cells treated terization of the changes in expression pattern associated
with the Kv1.3 blockers (Figure 6A). Similarly, the pro- with proliferation in in vivo and in vitro models of PM
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1210 Arterioscler Thromb Vasc Biol June 2010
showed a good correlation between both situations. Eigh- larization, proliferation was not affected if depolarization
teen of 22 genes that changed in the in vivo model were was induced with other channel blockers or with high
conservedinculturedVSMCs.Thelargernumberofgenes [K(cid:1)] .
e
that changed in cultured VSMCs can be attributed to their However, Kv1.3 inhibition could restrain hyperpolariz-
more homogeneous nature (a uniform population of syn- ing signals required for proliferation. This hypothesis is
thetic VSMCs), whereas endoluminal lesion samples are consistentwithourresultsshowinganattenuatedresponse
partially contaminated with endothelial cells, contractile to MgTx in high [K(cid:1)] .
e
VSMCs, and connective tissue. These contaminants could We have previously reported a similar association be-
also explain some of the changes present only in this tween Kv3.4 channel upregulation and proliferation in
model,asitismostlikelythecaseofSK3channels(small human uterine artery VSMCs,8 although in this prepara-
conductance K channels), whose expression is restricted tion, high [K(cid:1)] depolarization mimics proliferation inhi-
Ca e
to endothelial cells.9,24 Although specific changes induced bition by Kv3.4 blockade.30 Interestingly, the Kv1.3 gene
by the culture conditions cannot be excluded, our analysis is also upregulated in these cells,8 and Kv1.3 blockers
indicates that ion channel expression profile in cultured inhibit proliferation (Supplemental Figure VIB and VIC),
VSMCs reproduces reasonably well the changes in prolif- suggesting that the association between Kv1.3 upregula-
erating lesions in vivo. This observation is important tion and proliferation may be present in different vascular
beds.Infact,theionchannelexpressionprofileofcultured
consideringthetechnicallimitationsoftheinvivomodels
mesenteric VSMCs shows a remarkable similarity to that
for functional studies.
of femoral arteries (Supplemental Figure V), with Kv1.3
Only some of the channels highlighted here have been
being the predominant Kv1 channel gene and Kv1.3
previously reported as relevant to VSMC proliferation.
blockade inhibiting proliferation.
Downregulation of several Kv1 genes (Kv1.5, Kv1.2, and
Kv1.3 channels have been reported to associate with
Kv2.1) has been reported in other preparations.8,25 Also,
proliferation in T cells,17 endothelial cells,31 microglia,
decreased expression of Cav1.2 mRNA is consistent with
macrophages, oligodendrocyte progenitors, and carcinoma
the reported reduction in the functional expression of
L-typeCa2(cid:1)channelsinsyntheticVSMCs,5,26althoughwe cells,32,33 but this is the first description of their role in
VSMCproliferationandmigration.Inthisscenario,Kv1.3
didnotdetecttheconcomitantupregulationofgenesofthe
blockade could represent a new therapeutic approach to
Trpc and Cav3.x families described in these studies.
prevent unwanted remodeling.
Similarly, decreases of the BK channel genes (BK(cid:2)and
BK(cid:1)1 subunits) have been described,5,8,9,27 but we did not
Acknowledgments
find the associated increase in IK1 mRNA that was
We thank Esperanza Alonso for excellent technical assistance and
previously reported.5,9,10 Nevertheless, a contribution to theMerckCompanyforthegiftofcorreolide.
PM in our preparation of these channels (Trpc, Cav3.x, or
IK1) via posttranscriptional modulation cannot be ex- Sources of Funding
cluded. Overall, vascular bed variations may account for This work was supported by Ministerio de Sanidad, Instituto de
some of the recounted discrepancies, and it is tempting to Salud Carlos III grants R006/009 (Red Heracles), FS041139-0
(M.R.), and PI041044 (J.R.L.-L.); Ministerio de Educacio´n y
speculate that not only the expression profile of ion
CienciagrantsBFU2004-05551(M.T.P.-G.)andBFU2007-61524
channels but also the PM-induced changes could be (J.R.L.-L.); and Junta de Castilla y Leon grant GR242. Dr
vascular bed specific. In this context, the finding of Moreno-Domínguez is a fellow of the Spanish Ministerio de
conserved changes across different preparations is rele- Educacion y Ciencia.
vant, as it may reflect obligatory associations of certain
Disclosures
channels with vascular remodeling, representing novel
None.
therapeutic opportunities.
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