staurin
effect
cmicrovascularbarrierfunctionindiabeticanimalsandmonolayerbarrier
?cant
etal.,
ndothe
MicrovascularResearch80(2010)158–165
ContentslistsavailableatScienceDirect
Microvascular
endothelialbarrierdysfunction,whichcancausedisruptionofblood
vesselintegrity,hemorrhages,leukocyteadhesion,andtheformationof
smallthrombi(Yuanetal.,2000).Inearlystagesofdiabetes,injurytothe
endothelialbarriercommonlymanifestsasautoregulatorydysfunction
andincreasedendothelialpermeability(Youngetal.,2002;Scaliaetal.,
2007).Wehavepreviouslyshowninthecaseofmyocardiumischemia,
thesepathologicchangesmayincreaseparacellular?uxofinjuryfactors
andaggravatedamageofmyocardium(Wangetal.,2006;Gaoetal.,
2008).
Diabeticmicrovascularcomplicationscharacterizedbybarrierdys-
functionandpermeabilityincreasehavebeenlinkedtoactivationof
ProteinkinaseCs(PKCs)inanumberofstudies(Ishiietal.,1996;
Gutterman,2002;Kouroedovetal.,2004;Meieretal.,2007).PKCisa
PKCβIIisoform.RBXhasbeenshowntonormalizeendothelialfunction,
improverenalglomerular?ltrationrate,andpreventthelossofvisual
acuityindiabeticpatientssufferingfromretinopathy(Beckmanetal.,
2002;IdrisandDonnelly,2006;Mehtaetal.,2009).Whiletheeffectsof
PKC-βIIactivationandtheroleofRBXarewellunderstoodinrenaland
retinalmicrovascularcomplications,theyarestillnotclearincardiac
microvascularcomplications.Therefore,inthepresentstudyweaimto:
(i)determinewhetherPKC-βIIactivationandcardiacmicrovascular
barrierdysfunctionexistindiabeticratshearts;(ii)elucidatewhether
RBXcanreversemicrovascularbarrierdysfunctionandattenuatecell
junctiondamage;and(iii)investigatethemechanism(s)bywhichRBX
maymodulatecardiacmicrovascularinjuryinhighglucoseordiabetic
stateswithafocusonthePKC-βIIdependentsignaling.
serine/threoninekinasethatparticipatesince
diabeticvasculardamage(Yuan,2002;Budhiraja
etal.,2009).ActivationofProteinkinaseCs
?Correspondingauthors.
E-mailaddresses:wind8828@gmail.com(F.Cao),wanghc@fmmu.edu.cn
1
Contributedequallytothiswork.
0026-2862/$–seefrontmatter?2010ElsevierInc.All
doi:10.1016/j.mvr.2010.01.003
lialfunctionthatcanbe
disease.An
ovasculardysfunctionis
cularcomplicationsbothinvitroandinvivo(Ishiietal.,1996;Yuanetal.,
2000;AvignonandSultan,2006).
Ruboxistaurin(RBX)isaPKC-βinhibitorthatishighlyselectiveforthe
consideredthe?rststepintheprogressionofcardiovascular
importantunderlyingmechanismofdiabeticmicr
Cardiacmicrovascularendothelialcells
(CMECs)
PKC-βinhibitor
Permeability
Introduction
Cardiovascularcomplicationsaresigni
mortalityinthehyperglycemia(Capes
(DM)isassociatedwithchangesine
endothelialbarrierdysfunction.PKC-βinhibitorRBXpreventedchroniccardiacmicrovascularbarrier
dysfunctionandimprovedendothelialcell–celljunctionalfunctioninhighglucosestates.
?2010ElsevierInc.Allrightsreserved.
causesofmorbidityand
2000).Diabetesmellitus
membranousprotein(Xuetal.,2008).Inparticular,theβisoformofPKC
(PKCβIandPKCβII)isexpressedincardiovasculartissuesandactivated
bycirculatinghighglucoseandfattyacids.Recently,severalreportshave
demonstratedthatPKC-βIIplaysasigni?cantroleindiabeticmicrovas-
llsignaltransductionin
andSingh,2008;Mehta
iselevatedexpressionin
Materialsandmethods
Animalpreparation
Allanimalex
InstitutesofHealth
(H.Wang).
rightsreserved.
protectivepropertiesofPKC-βinhibitoragainstcardiac
Diabetes
functionofculturedcardiacmicrovascularendothelialcells(CMECs),reproducingthesameeffectasPKC-βII
siRNA.Theseresultsprovidenewinsightinto
Keywords:
treatmentprotectedcardia
RegularArticle
APKC-βinhibitortreatmentreversescardiac
diabeticrats
LipingWei
a,b,1
,ZhiyongYin
a,1
,YuanYuan
a,1
,Andrew
ChunxiaDi
a
,RongqingZhang
a
,FengCao
a,
?,Haichang
a
DepartmentofCardiology,XijingHospital,FourthMilitaryMedicalUniversity,Xi''an,710032,
b
DepartmentofCardiology,Tianjinunionmedicinecenter,Tianjin,300121,China
c
DepartmentofRadiology,MolecularImagingProgramofStanford,StanfordUniversity,Palo
abstractarticleinfo
Articlehistory:
Received6September2009
Revised16December2009
Accepted5January2010
Availableonline14January2010
ThePKC-βinhibitorruboxi
complications.However,the
thisstudy,weaimedtoinvestigate
barrierdysfunctioninhighglucose
PKC-βIIactivationandphospho
journalhomepage:www.el
microvascularbarrierdysfunctionin
Hwang
c
,AndrewLee
c
,DongdongSun
a
,FeiLi
a
,
Wang
a,
?
Shaanxi,China
Alto,CA,USA
(RBXorLY333531)preventsdiabeticrenalandretinalmicrovascular
ofRBXondiabeticcardiacmicrovasculardysfunctionisstillunclear.In
theeffectsandmechanismsofRBXtreatmentuponcardiacendothelial
states.WedemonstratedRBXtreatmentsuppressedhighglucoseinduced
rylationofβ-catenininvivoandinvitroexperiments.Meanwhile,RBX
Research
sevier.com/locate/ymvre
forinvivoexperiments
perimentswereperformedundertheNational
GuidelinesontheUseofLaboratoryAnimals
159L.Weietal./MicrovascularResearch80(2010)158–165
andwereapprovedbytheFourthMilitaryMedicalUniversity
CommitteeonAnimalCare.30maleSprague–Dawleyrats(weight
250–280g)wereinducedtoadiabeticstatebyasingleintraperitoneal
injectionofstreptozotocin(STZ,50mg/kgin0.9%saline,Sigma,USA).
Bloodglucoselevelsweremeasured1weekafterSTZinjection.
Animalswithglucoselevelsequaltoorgreaterthan300mg/dlwere
classi?edasdiabetic(Talpuretal.,2005).Diabeticratsweredivided
into3groups:(1)DMgroup(n=10)thatdidnotreceivetreatment;
(2)RBXgroup(n=10)thatreceiveddailytreatmentsofruboxistaurin
(Lilly,USA)at5mg/kgofbodyweightfor2weeks;(3)Vehiclegroup
(n=10)thatreceivedonlysalineonadailybasisfor2weeks.Age-
matchednormalrats(n=10)wereusedasnon-diabeticcontrols.
Examinationofbarrierfunctionofcardiacmicrovascularendothelial
cells(CMECs)viatransmissionelectronmicroscope
Totestforintegrityofendothelialcelltocelljunctions,heartsof
anaesthetizedratswereperfusedviathecarotidarterywith50mlof
pre?xativesolution(100mMTris,pH7.2,150mMNaCl,5.6mMKCl,
1mMMgCl
2
,2.5mMCaCl
2
,3.7mMglucoseand3.6mMprocaine)
followedby250mlof?xingsolution(2.5%glutaraldehydeand2%
paraformaldehydein0.1Msodiumcacodylatebuffer,pH7.2,
containing2%lanthanumnitrate)usingaperistalticpump.The
perfusionpressurewasmonitoredwithamercurymanometerand
neverexceeded100mmHg.Afterperfusion,thehearttissuewaskept
inthesame?xativewithoutlanthanumfor1h.Thesampleswere
thenrinsedinwashingsolution(0.15MNaClplus0.2Msucrose).
Ultra-thinsections(60nmthick)werecut,mountedoncoppergrids
(200mesh)andexaminedusingatransmissionelectronmicroscope
tocheckforthepresenceoflanthanumnitrate(JEM-2000EX,Japan).
ExaminationofintegrityofCMECsviascanningelectronmicroscope
After?xing,tissuesampleswereexaminedviascanningelectron
microscopytoassessmicrovascularendothelialintegritywithinthe
vesselwallofdiabeticandcontrolrathearts.Animalheartsreceived
10mlMercoxresin(resin20ml,catalyst0.5mg)(SPI,USA)at
perfusionpressurenomorethan100mmHgthroughtheaortaand
weredippedinasolutionconsistingof5%sodiumhydroxidesolution
(Verlietal.,2008).Afterremovalofallconnectivetissuecomponents
surroundingthebloodvessels,regularpretreatmentsinaccordance
withstandardinstructionwereemployed,includingdehydration,
desiccationandgilding.Preparedsamplesweresubsequentlyexam-
inedwithascanningelectronmicroscope(HITACHIS-3400N,Japan).
IsolationandmaintenanceofCMECsforinvitroexperiments
CMECswereisolatedfrom15maleSprague–Dawleyratsweighting
100–120g.Leftventriclesofratswereharvestedandmincedinto1mm
3
smallpiecesafterremovaloftheendocardialendotheliumandthe
epicardialcoronaryarteries.ThepieceswereincubatedincollagenaseII
(1mg/ml,Invitrogen,USA)andsubsequentlyculturedinEndothelial
GrowthMediumconsistingofde?nedgrowthfactorsandsupplemented
withadditionalFBSupto15%ofthe?nalconcentration(Hendrickxetal.,
2004).Passage3–5cellswereusedforfurtherstudies.RBX(Alexis,USA)
dissolvedindimethylsulfoxide(DMSO,Sigma,USA)toa?nal
concentrationof1,10,or100nmol/L.Mediawerereplacedtodifferent
conditionsaftercon?uence:normalglucosemedium(5.5mmol/L),high
glucosemedium(25mmol/L),highglucoseplusRBX-1(1nmol/L),high
glucoseplusRBX-2(10nmol/L),highglucoseplusRBX-3(100nmol/L),
andhighglucoseplusvehicle(DMSO).
Measurementoftransendothelialelectricalresistance
Transendothelialelectricalresistance(TER)isameasureofthe
ionicconductanceofendothelialcellsandcanbeusedtoassess
junctionalfunction.TERincreaseswhenendothelialcellsadhereand
spreadout,anddecreaseswhenendothelialcellsretractorlose
adhesion.Anelectricalendothelialresistancesystem(Millipore,USA)
wasusedtomeasureTERaspreviouslydescribedindetail(Chiang
etal.,2009).Con?uenceofastandardCMECsmonolayerwasassessed
asastabilizedbasalresistanceof1000Ω.Aftercon?uence,the
mediumwasreplacedbynormalmedium(5.5mmol/L),highglucose
medium(25mmol/L),highglucosemediumplusRBX(1,10,
100nmol/L)orhighglucoseplusvehicle.TERindifferentmedia
wasmeasuredafterincubationfor24h.Thesemeasurementsprovide
ahighlysensitivebiophysicalassayofcelljunctionalfunction.This
procedurewasrepeated?vetimes.
QuantitativeassayofCMECs''permeability
FITC-Dextranclearancewasmeasuredtoassesschangesin
endothelialpermeability.CMECsat100,000cells/insertwereseeded
ontocollagencoatedinsertofanInVitroVascularPermeabilityAssay
kit(ECM640,Millipore,USA)andgrowntocon?uence.After
con?uence,theculturedmediumwasalsoreplacedbydifferent
mediadescribedabove.Afterincubationfor24hinthesemedia,FITC-
Dextran(?nalconcentration1mg/mL)wasaddedtothemediumand
allowedtopermeatethroughthecellmonolayer.Theextentof
permeabilityafter2hwasdeterminedbyusinga?uorescentplate
reader(Bio-Rad,USA)tomeasuretheFITCcontentremainingonthe
plate.Thisprocedurewasrepeated?vetimes.
Immuno?uorescencestainingofPKC-βII
InordertolocalizePKC-βIIexpressionwithinthediabeticheartand
culturedCMECs,immuno?uorescentstainingwasperformed.Heart
tissuewaspreparedaspreviouslydescribed(Yoonetal.,2005).Tissues
were?xedwithparaformaldehydeandsectionedinto4to5μmslides.
CMECswerewashedoncewithPBS,?xedin4%paraformaldehydefor
10min,washedagainwithPBS,andpermeabilizedwith0.2%Triton100
for5min.HearttissueandCMECswereincubatedovernightwithanti-
PKC-βIIantibody(1:200,Abcam,British).Afterwards,slidesandcells
werewashed3timeswithPBSandincubatedwithsecondaryFITC-
labeledantibody(ZhongshanCO,China)for1h.AnIX71cofocal
microscope(Olympus,Japan)wasusedtoassessimages.
WesternblotanalysisforPKC-βII,phosphor-β-cateninandVE-cadherin
InordertoexaminetheactivatedPKC-βIIthatwastranslocatedfrom
cellcytoplasmtomembrane.Membranousproteinfromhearttissue
andculturedCMECswasisolatedusingtheEukaryoticMembrane
ProteinExtractionReagentKit(Pierce,USA).Monoclonalantibody
againstPKC-βII(1:1000,Abcam,British),phosphor-β-catenin(1:1000,
SantaCruz,USA),andVE-cadherin(1:1000,Abcam,British)wereused
forwesternblotanalysis.Nitrocellulosemembraneswereincubated
withHRP-conjugatedanti-mouseimmunoglobulinGantibody(1:2000,
CellSignaling,USA)for1handtheblotwasdevelopedwitha
Supersignalchemiluminescencedetectionkit(Pierce,USA).Immuno-
blottingwasvisualizedwithanImageStation400(Kodak,Japan).
TransfectionofPKC-βIISiRNAintoCMECs
TransfectionswereperformedwithEntransterTM-R(EngreenBio-
system,China)accordingtothemanufacturer''sinstructions.PKC-βIIand
GAPDH-speci?csiRNAsweresynthesized(SantaCruz,USA)andusedfor
transfection.ThesenseandantisensestrandsofthePKC-βIIsiRNAwere
GUCAGAUGCUGAUGUUCCUUUandUUCAGUCUACGACUACAAGGA.
CMECsweretransfectedafter48hofculture.EntransterTMR(8μL)
wasaddedto100μLofOptiMEMserum-freemediumcontaining2nmol/
LofeachsiRNAoligo,incubatedfor10min,andaddedtothe6-cmplate
containing2mlmedium.GAPDHsiRNAwasusedasanegativecontrol.
TheFITC-labeled(SantaCruz)controlsiRNAwasusedasamarkerof
transfectionef?ciency.TERandpermeabilityacrosscon?uentCMECs
monolayerswereperformedasdescribedabove.Theef?cacyofPKC-βII
silencingandreductionsinproteinexpressionwasassessedafter72hin
thesamegroupofcellsusingWesternblotanalysis.
Statisticalanalysis
Allvaluesarepresentedasmean±SD(ofnindependent
experiments).Alldata(exceptWesternblotdensity)werecompared
byANOVAfollowedbyBonferronicorrectionforposthocanalysis.P
valuesb0.05wereconsideredstatisticallysigni?cant.Allstatistical
testswereperformedusingGraphPadPrismsoftwareversion4.0
(GraphPadSoftware,SanDiego,CA).GraphofTERwasperformed
usingSigmaplot8.0software(SPSSInc,Chicago,USA).
Results
RBXtreatmentprotectscardiacmicrovascularbarrierfunctionin
diabeticrats
Lanthanumnitrateisareagentthathasbeenusedinanumberof
studiestotestbarrierfunctionofendothelialcells(Raposoetal.,
2007).Underhealthyconditions,lanthanumnitrateisunableto
traverseendothelialcelltocelljunctionsinsigni?cantquantities,but
whensuchbarriersarede?cientasinpathologicconditionsitmay
crosscellboundariesviaparacellulardiffusion.Inthisstudy,
applicationoflanthanumnitraterevealedsigni?cantde?citsin
barrierintegrityofdiabeticanimalhearts.Ascomparedtocontrol
animalsinwhichpresenceoflanthanumnitratewasregulatedtothe
bloodvessellumen(Fig.1A),lanthanumnitratewasfoundtodiffuse
acrossvessellumentothebasallaminaindiabeticanimals(Fig.1B).
Diffusionoflanthanumnitrateacrossendothelialcellswasattenuated
byadministrationofRBX,showingthatRBXiscapableofprotecting
cardiacendotheliumfromdamageinducedbydiabetes(Fig.1C).
Animalsthatreceivedtreatmentofvehiclealoneexhibitedlossof
barrierfunctioncomparabletodiabeticanimals(Fig.1D).
RBXtreatmentmaintainsCMECsintegrityindiabeticrats
ScanningelectronmicroscopyrevealedthesurfaceofCMECsin
controlanimalstobesmoothandwell-integrated(Fig.1EandI).In
contrast,CMECsindiabeticanimalswereobservedtobehighly
irregular,withnumerousexvaginationsandinvaginations.Cell
junctionintegritywaslargelycompromisedandcellswerenotwell
integratedwiththevessel(Fig.1FandJ).RBXtreatmentattenuated
changes,preservingCMECsintegrityandvesselintegration(Fig.1G
andK).Animalsthatreceivedtreatmentofvehiclealonedidnot
exhibitimprovement(Fig.1HandL).
DecreasedPKC-βIIactivatedandphosphor-β-catenincontributeto
endothelialbarrierrepair
Immuno?uorescentstainingshowedPKC-βIIactivatedexpression
tobelowinheartsofcontrolanimals(Fig.2A)buthighlyelevatedin
thediabeticgroup(Fig.2B).TreatmentwithRBXdownregulatedPKC-
and
solution
ultrastructure
animals
attenuated
that
and
integrity
160L.Weietal./MicrovascularResearch80(2010)158–165
Fig.1.Cardiacmicrovascularpermeabilityobservedbytransmissionelectronmicroscope
heartsofanaesthetizedratswereperfusedviathecarotidarterywith50mlofpre?xative
cardiacmicrovascularpermeabilityvialanthanumnitratetracerandtoexamineendothelial
witharrows.Lanthanumnitratewasonlyregulatedtothebloodvessellumenincontrol
laminaindiabeticanimals(B).Diffusionoflanthanumnitrateacrossendothelialcellswas
anddiffusionoflanthanumnitrateacrossendothelialcells(D).Thisresulthasshown
Scanningelectronmicroscopywasusedtoanalyzechangesinmorphologyofcardiacmicrovessels.
methods.ThesurfaceofCMECsincontrolanimalswassmoothandwell-integrated(E
numerousexvaginationsandinvaginations(FandJ).CMECsinRBXtreatmentexhibited
displayedirregularandrough(HandL).
scanelectronmicroscope.Totestforintegrityofendothelialcelltocelljunctions,
containinglanthanumnitrate.Transmissionelectronmicroscopywasusedtoassay
(scalebar:2μm).Theextracellulartracerlanthanumnitrateishighlighted
(A),andlanthanumnitratewasfoundtodiffuseacrossvessellumentothebasal
inRBXtreatment(C).Vehicletreatmentaloneexhibitedlossofbarrierfunction
RBXiscapableofprotectingcardiacendotheliumfromdamageinducedbydiabetes.
AnimalheartsreceivedperfusionofMercoxresinasdescribedinMaterialsand
I).Incontrast,CMECsindiabeticanimalswereobservedtobehighlyirregular,with
andvesselintegration(GandK).Animalsthatreceivedtreatmentofvehicle
?uo
gsh
pre
an
min
ead
161L.Weietal./MicrovascularResearch80(2010)158–165
Fig.2.PKC-βII,phosphor-β-cateninandVE-cadherinexpressioninhearttissue.Immuno
methods.(Highlightedwitharrows,AtoD,scalebar:20μm).Immuno?uorescentstainin
elevatedinthediabeticgroup(B).TreatmentwithRBXdownregulatedPKC-βIIactivatedex
affectPKC-βIIactivatedexpression(D).WesternblotanalysisofPKC-βII,phosphor-β-catenin
antibodyshowPKC-βIIexpressionwasincreasedindiabeticsanddown-regulatedbythead
showphosphor-β-cateninexpressionwasincreasedindiabeticsanddown-regulatedbyth
βIIactivatedexpressioninanimalswithdiabetes(Fig.2C).Admin-
istrationofthevehiclealonedidnotsigni?cantlyaffectPKC-βII
activatedexpression(Fig.2D).Westernblotsemi-quantization
revealedexpressionofPKC-βIIactivatedandphosphor-β-cateninto
besigni?cantlyelevatedindiabeticheartsascomparedtonon-
diabeticcontrols(1.06±0.09vs.0.76±0.06forPKC-βII;0.74±0.13
vs.0.47±0.11forphosphor-β-cateninin,Pb0.01)(Fig.2E).RBX
treatmentsigni?cantlyloweredlevelsofPKC-βIIactivated(0.97±
0.11vs.0.78±0.10,Pb0.05)andphosphor-β-catenin(0.80±0.11vs.
0.59±0.07,Pb0.05)(Fig.2F).VE-cadherinexpressiondidnotdiffer
amongthevariousgroups(Fig.2G).
RBXpretreatmentprotectsmonolayerbarrierfunctionofCMECs
MeasurementofTERisanaccuratemethodofassessing
junctionalintegrityforendothelialcells.AhighTERisassociated
withrelativelyimpermeablejunctionsofanendothelialmonolayer,
whilealowTERindicateselevatedpermeability(Fig.3A).Inthis
study,CMECsculturedinhighglucosemediumhadsigni?cantly
lowerTERthanthoseculturedinnormalglucosemedium(760.3±
72.1Ωvs.1053.2±76.3Ω,Pb0.01).AdditionofRBXtohighglucose
mediumatconcentrationsof10nmol/Land100nmol/Linduced
endothelialbarrierenhancementasdetectedbyincreasedTER
comparedwithvehicleonlytreatment(1079.2±72.8Ωvs.810.3±
71.6Ω,Pb0.01,905.6±77.9Ωvs.810.3±71.6Ω,Pb0.05,
respectively).AdditionoflowconcentrationsofRBX(1nmol/L)to
highglucosemediumdidnotaffectTERsigni?cantly(Fig.3B).
TocomplementandvalidateTERresults,permeabilityofthe
monolayerwasalsoassessedviatheadditionofaFITC-Dextransolution
totheCMECsmonolayerfollowedby?uorescenceanalysis.FITC-
DextranwasaddedtoCMECsculturedundernormalandhighglucose
conditions.Relative?uorescenceunit(RFU)inthehighglucosemedium
group).Analysisofmembraneswithanti-VE-cadherinantibodyshowVE-cadherinexpression
rescencestainingofPKC-βIIinhearttissuewasdetectedasdescribedinMaterialsand
owedPKC-βIIactivatedexpressiontobelowinheartsofcontrolanimals(A)buthighly
ssioninanimalswithdiabetes(C).Administrationofthevehiclealonedidnotsigni?cantly
dVE-cadherinexpressioninhearttissue(EtoG).Analysisofmembraneswithanti-PKC-βII
istrationofRBX(E).Analysisofwholehomogenatewithanti-phosphor-β-cateninantibody
ministrationofRBX(F)(??Pb0.01vs.Non-diabeticcontrol,#Pb0.05vs.Vehicletreatment
wasmuchhigherthaninthenormalmedium(394.5±36.9RFUvs.
279.1±14.9RFU,Pb0.01).RBXpretreatmentreducedFITC-dextran
clearanceatconcentrationsofRBXat10nmol/L(369.9±25.9RFUvs.
282.5±24.2RFU,Pb0.01)andat100nmol/L(369.9±25.9RFUvs.
325.7±20.5RFU,Pb0.05),suggestingthatRBXataconcentrationofat
least10nmol/Lsigni?cantlyenhancesendothelialbarrierfunctionby
reducingleakage(Fig.3C).
RBXpretreatmentnormalizesexpressionofPKC-βIIand
phosphor-β-catenininvitro
Notably,PKC-βII(1.17±0.13vs.0.71±0.07,Pb0.01)andphos-
phor-β-catenin(0.92±0.11vs.0.56±0.07,Pb0.01)expressionwas
markedlyhigherinCMECsculturedinthehighglucosemediumthan
inthenormalglucosemedium.PKC-βIIandphosphor-β-catenin
expressioninthehighglucosegroupwerereversedbypretreatment
withRBXat10nmol/L(1.13±0.12vs.0.67±0.14forPKC-βII,
Pb0.01)(0.76±0.07vs.0.47±0.06forphosphorylationofβ-catenin,
Pb0.01)andat100nmol/L(1.13±0.12vs.0.83±0.07forPKC-βII,
Pb0.05)(0.76±0.07vs.0.62±0.09forphosphorylationofβ-catenin,
Pb0.05)comparedwithvehiclepretreatment(Fig.4AandB).In
contrast,VE-cadherinexpressiondidnotdifferbetweenthehigh
glucosemediumandthehighglucosemediumpretreatedwithRBX
(Fig.4C).
EffectofPKC-βIIsiRNAonPKC-βII,phosphor-β-cateninandVE-cadherin
expression
TheeffectsofRBX(10nmol/L)onproteinexpressionofphosphor-
β-cateninandVE-cadherinwascomparedwithuseofPKC-βIIsiRNA.
HighglucosemediumresultedinupregulationofPKC-βIIexpression
andtheadditionofGAPDHcontroldidnotinhibitPKC-βIIactivation.
didnotdifferamongthevariousgroups(G).
Fig.3.EffectsofRBXonpermeabilityofCMECsmonolayer.CMECsculturedongoldmicroelectrodesandsubjectedtotransendothelialelectricalresistance(TER)measurementsto
detectchangesinbarrierpermeability.Increasesinresistancecorrespondtotighteningofjunctionaladhesionrelatedwithenhancedbarrierfunction,whiledecreasesinresistance
correspondtobarrierdysfunctionandincreasedparacellularpermeability.Trans-endothelialelectricalresistance(TER)overtimeinnormal,highglucose,highglucoseplus
treatment,andhighglucoseplusvehiclegroups.TERofCMECsmonolayerwaspresenteddifferenttendencyindifferentgroup,whereaspretreatmentwithRBXinhibiteddecreaseof
TERandenhancedbarrierfunction(A).RBXattenuatesthedecreaseinjunctionalintegrityafterincubationfor24hasmeasuredbyTER(B).RBXattenuatesthepermeabilityincrease
ofmonolayerCMECsafterincubationfor24hasmeasuredbyFITC-Dextranclearance(C).(##Pb0.01vs.Nor,??Pb0.01vs.HG+Vehicle,?Pb0.05vs.HG+Vehicle)Nor:normal
glucosemedium,HG:highglucosemedium,HG+Vehicle:highglucoseplusvehiclemedium.
Fig.4.PKC-βII,phosphor-β-cateninandVE-cadherinexpressioninCMECsinvitro.PKC-βIIactivatedandVE-cadherinwereexaminedusingcellmembranousproteinandβ-catenin
phosphorylationwasexaminedusingwholehomogenate.PKC-βIIwasactivatedinCMECswithhighglucosemediumanddown-regulatedbytheadministrationofRBX(A).
Phosphor-β-cateninexpressionincreasedinthehighglucosemediumandwassigni?cantlydownregulatedbyadministrationRBX(B).VE-cadherinexpressioninvariousgroupsdid
notdiffersigni?cantly(C).(##Pb0.01vs.Nor,??Pb0.01vs.HG+Vehicle,?Pb0.05vs.HG+Vehicle)Nor:normalglucosemedium,HG:highglucosemedium,HG+Vehicle:high
glucoseplusvehiclemedium.
162L.Weietal./MicrovascularResearch80(2010)158–165
Bycomparison,useofRBXandPKC-βIIsiRNAsigni?cantlyinhibited
activatedPKC-βIIexpression(Fig.5AtoD).PKC-βIIandphosphor-β-
cateninexpressioninthehighglucosemediumwerereversedby
pretreatmentwithRBX(0.59±0.08forPKC-βII,Pb0.01;0.56±0.09
forphosphor-β-catenin,Pb0.01)andPKC-βIIsiRNA(0.51±0.13for
PKC-βII,Pb0.01;0.62±0.11forphosphor-β-catenin,Pb0.01).There
werenosigni?cantdifferencesbetweenRBXtreatmentanduseof
PKC-βIIsiRNA(Fig.5EandF).Incontrasttophosphor-β-catenin,
levelsofVE-cadherindidnotchangeafterexposureofGAPDHcontrol,
RBX,andPKC-βIIsiRNA(Fig.5G).
EffectofPKC-βIIsiRNAonbarrierfunctionofCMECsmonolayer
AdditionofPKC-βIIsiRNAtoCMECsresultedinendothelialbarrier
enhancementasevidencedbyanincreasedTER(1025.4±87.7Ωvs.
755.6±75.9Ω,Pb0.01)anddecreasedpermeability(385.3±27.3
RFUvs.284.3±31.7RFU,Pb0.01).Therewerenosigni?cant
differencesbetweenuseofPKC-βIIsiRNAandRBX.BothPKC-βII
siRNAandRBXselectivelyinhibitedactivatedPKC-βIIexpressionand
protectedendothelialbarrierfunction(Fig.6).
Discussion
Vascularendothelialcellsplayamajorroleinmaintaining
cardiovascularhomeostasisinnormalconditions.Diabetesmellitus
substantiallyimpairsthepropertiesoftheendotheliumandleadsto
endothelialdysfunction,especiallyphysicalbarrierfunction,which
canbeconsideredthecrucialprogressionofcardiovasculardisease
Yuanetal.(2000)havereportedasigni?canttranslocationofboth
PKC-βIIand-?isoformsfromthecytosoltothemembraneinthe
heartsofdiabeticanimalsaccompanyingwithbasalpermeabilityto
andVE-cadherininCMECsinvitro.CMECsweretransfectedwithPKC-βIIsiRNAorcontrol
?uorescenceinCMECs(AtoD,scalebar:40μm).Immuno?uorescentstainingshowed
elevatedinthehighglucose(B).TreatmentwithRBXdownregulatedPKC-βIIactivated
βII,phosphor-β-cateninandVE-cadherinexpressioninCMECs.TransfectionwithPKC-βII
didnotchangetheexpressionofVE-cadherin(EtoG).PKC-βIIdepletioninducedbyPKC-βII
thesameeffectasPKC-βIIsilencingreducedproteinexpressionofPKC-βIIand
Fig.6.DepletionofPKC-βIIenhancedbarrierfunctionofCMECsmonolayerculturedin
highglucose.EffectsonbarrierfunctionofCMECstransfectedwith8nMPKC-βIIsiRNA
orcontrolsiRNAcomparedwitheffectsofRBXat10nmol/L.After24h,TERofCMECs
monolayerwasmeasuredbyanelectricalendothelialresistancesystem(Millipore,
USA)andFITC-Dextranclearancewasmeasuredtoassesschangesinendothelial
163L.Weietal./MicrovascularResearch80(2010)158–165
(Avogaroetal.,2006;Yuanetal.,2007).Severalstudieshave
discussedthemolecularmechanismsunderlyingendothelialdys-
function(WautierandSchmidt,2004;IdrisandDonnelly,2006;
Mohamadinetal.,2007)andtheactivationofPKCsorPKCsisoforms
byhyperglycemiahasbeenfoundtobethemostimportant(Das
EvcimenandKing,2007).
TheindividualisoformsofPKCsresponddifferentlytohypergly-
caemia,however,bothexperimentsinvivoandinvitrosupportthe
predominantroleofPKC-βIIindiabeticmicrovascularcomplications.
Fig.5.EffectsofPKC-βIIdepletiononproteinexpressionofPKC-βII,phosphor-β-catenin
siRNAasdescribedinMaterialsandmethods.PKC-βIIexpressionwasdetectedbyimmuno
PKC-βIIactivatedexpressedtobelowinCMECsculturedinnormalmedium,(A)buthighly
expression(C)similarwiththeeffectsofPKC-βIIsiRNA(D).WesternblotanalysisofPKC-
speci?csiRNAin?uencedtheproteinexpressionofPKC-βIIandphosphor-β-catenin,but
speci?csiRNAwascon?rmedbyWesternblot.ThepharmacologicalinhibitorRBXreproduced
phosphor-β-catenin.Representativeblotsof5independentexperimentsareshown(??Pb0.01
permeability.Trans-endothelialelectricalresistance(TER)overtimeinGAPDHcontrol,
RBXtreatment,andPKC-βIIsiRNAgroups(A).PretreatmentwithRBXinhibited
decreaseofTERandenhancedbarrierfunctionsimilarwiththeeffectofPKC-βIIsiRNA
(B).RBXattenuatesthepermeabilityincreaseofCMECsmonolayersimilarwiththe
effectofPKC-βIIsiRNA(C)(??Pb0.01vs.GAPDHcontrol,##Pb0.01vs.GAPDHcontrol).
vs.GAPDHcontrol,##Pb0.01vs.GAPDHcontrol).
164L.Weietal./MicrovascularResearch80(2010)158–165
albuminelevatedincoronaryvenues.Similarly,Beckmanetal.(2002)
alsoshowedthathyperglycemiaimpairsendothelialfunctionpartly
viaPKCβactivation.AlthoughthesereportsdemonstratePKC-βII
activationasthedirectlinkbetweenhyperglycemiaandcardiac
vascularendothelialdysfunction,however,theeffectandmechanisms
ofPKC-βIIactivationoncardiacmicrovascularfunctionislessclear.
Inthepresentstudy,weappliedanidealandhighselectivedrug
RBXtodemonstratethePKC-βIIisoformactivationandsubsequent
cardiacmicrovasculardamageinresponsetohyperglycemiainvivo
andinvitro.
Inordertotestendothelialpermeabilityinvivo,lanthanumnitrate
permeabilityassayswereperformed.Lanthanumnitratehasa
moleculardiameterof4nmandisunabletopassthroughcell
junctionsundernormalconditions.Underconditionswheremicro-
vascularendothelialintegrityiscompromised,however,itisableto
diffuseparacellularlyacrosscelljunctions(Raposoetal.,2007).Our
resultsshowthatinheartsofdiabeticratswithPKC-βIIactivation,
cardiacmicrovesselshaveincreasedpermeabilitytolanthanum
nitrate,indicatingbarrierdysfunction.Ameliorationofcell–cell
junctionalimpairmentfollowingRBXadministrationdemonstrates
thatPKC-βIIactivationplaysanimportantroleinthispathological
process.Throughscanningelectronmicroscopy,wealsofoundthat
CMECsindiabeticanimalshaveabnormalvascularstructureand
compromisedintegrityofendothelialcelljunctions.RBXtreatment
reversedthesepathologicalchangesaswell.
Andwecon?rmedtheprotectiveeffectsofRBXonCMECs
monolayerbyassayingcellpermeabilityandTER.Wefoundthatat
aconcentrationof10nmol/L,RBXexertsamaximallybarrierfunction
protectiveroleandPKC-βIIexpressioninhibition.Interestingly,
barrierfunctionwasnotenhancedathighdosesofRBXsuchas
100nmol/L.ThereasonforthismaybeduetoinhibitionofotherPKC
isoformsathighconcentrationsofRBX,whichmayexertadditional
adverseeffectstoendothelialcells.Resultsofthesetestsindicated
prolongedculturedCMECsunderhighglucoseconditionscaninduce
PKC-βIIactivationandCMECsmonolayerbarrierdysfunctionwhile
pretreatmentwithRBXcanattenuatethesechanges.Wecompared
theeffectsofRBXat10nmol/LwiththetransfectionofPKC-βIIsiRNA
intoCMECs.WefoundRBXselectivelyinhibitedPKC-βIIactivated
expressionandprotectedendothelialbarrierfunction,reproducing
thesameeffectasPKC-βIIsiRNA,whichfurthersupportsour
conclusionthatRBXcontributesprotectiveeffectsuponmicrovascu-
latureviadownregulationofPKC-βIIandnotindependent
mechanisms.
Endothelialbarrierfunctionisdeterminedbyabalancebetween
adhesiveforcesatintercellularjunctionsandactingenerated
centripetalforces.Lossofadhesiveforceatthejunctionsand
increasedactingeneratedforcemayincreaseparacellular?uxof
?uidsandmacromolecules(BaldwinandThurston,2001;Bazzoniand
Dejana,2001).Cadherinsbinddirectlytoβ-cateninandtop120.
Linkageofthecadherinsviacateninstotheactincytoskeletonisthe
mechanismbywhichcateninsstrengthencadherin-mediatedadhe-
siveforce(Dejana,2004;Guoetal.,2008;Vestweber,2008).VE-
cadherinishighlyexpressedinmicrovascularendothelialcells,which
isaprimarycomponentoftheadherentjunctionproteinthat
maintainsadhesiveforce(Orlovaetal.,2006;Dejanaetal.,2008).
β-cateninisanimportantproteininsignaltransductionofjunction–
cytoskeletoninteractions(Aberleetal.,1996;BazzoniandDejana,
2001).Exogenousorendogenousagentsmayaffectthestabilityof
thissystembyinducingphosphorylationand/ordistributionof
junctionproteins(Coradaetal.,2001).Manystudies(Tinsleyetal.,
1999;Shasbyetal.,2002;Yuan,2002)havedemonstratedhyperper-
meabilityfactorsstimulateβ-cateninphosphorylationandsimulta-
neouslycauseendothelialgapformation.Weshowedinhibitionof
PKC-βIIanddownregulationofβ-cateninphosphorylationpreferen-
tiallyprotectagainstCMECsmonolayerbarrierdysfunction.PKCβII
activationwasparalleledwithincreasesinphosphorylatedβ-catenin,
indicatingthatβ-cateninisdirectlyphosphorylatedbyactivatedPKC
βIIorbyaPKC-dependentactivationofanotherkinase(Mehtaetal.,
2001).Phosphor-β-cateninwasalsofoundtobeelevatedunderhigh
glucoseanddiabeticconditionswhereasVE-cadherinexpressionwas
maintainedatconstantlevelsregardlessoftheamountofglucosein
thecultureenvironment.Weproposethatphosphor-β-catenin
inducedbyPKC-βIIactivationpromotesthedissociationofVE-
cadherinfromVE-cadherin–catenincomplexandleadstodeteriora-
tionofthefundamentalstructureofnormalCMECsbarrierfunction.
RearrangementofVE-cadherinnotVE-cadherinphosphorylation
modulatedbyPKC-βIIactivationmostlikelycontributestolossof
adhesiveforce.Thesehypothesesaresupportedbyotherstudiesthat
showmultipleantibodiesdirectedtotheextracellulardomainofVE-
cadherincandisruptendothelialbarrierfunctioninvitro(Corada
etal.,2001;Londonetal.,2009).
Takentogether,Ourdatafurtherdemonstratethatβ-catenin
phosphorylationwasanimportantsignalresponsiblefordiminishing
celltocellcontactandbreakingdownthecardiacbarrierfunctionin
diabeteswithPKC-βIIactivation.RBXpretreatmentreducedcardiac
microvascularbarrierimpairmentandimprovedendothelialcellular
junctioninvivoandinvitro.Themechanismsresponsibleforthese
effectsofRBXmightbepartlymediatedbyPKC-βIIactivationand
phosphor-β-cateninsignalpathway.
Con?ictofintereststatement
Thereisnocon?ictofinteresttodeclare.
Acknowledgments
ThisstudywassupportedbygrantsfromtheNationalNatural
ScienceFoundationofChina(NSFC,No.30770784,30670969)and
XijingResearchBoostingProgram(FC,No.XJZT07Z05,XJZT08Z04).
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