2024年4月6日发(作者：偶昭懿)
AngiogenicFactorAGGF1PromotesTherapeutic
AngiogenesisinaMouseLimbIschemiaModel
QiulunLu
1
,YihongYao
1
,YufengYao
1
,ShizhiLiu
1
,YuanHuang
1
,ShanLu
1
,YingBai
1
,BishengZhou
1
,
YanXu
1
,LeiLi
1
,NanWang
1
,LiWang
1
,JieZhang
1
,XiangCheng
2
,GangjianQin
3
,WeiMa
4
,ChengqiXu
1
,
XinTu
1
*,QingWang
1,5
*
1KeyLaboratoryofMolecularBiophysicsoftheMinistryofEducation,CollegeofLifeScienceandTechnology,CenterforHumanGenomeResearch,Cardio-XInstitute,
HuazhongUniversityofScienceandTechnology,Wuhan,People’sRepublicofChina,2InstituteofCardiology,UnionHospital,TongjiMedicalCollege,Huazhong
UniversityofScienceandTechnology,Wuhan,People’sRepublicofChina,3FeinbergCardiovascularResearchInstitute,NorthwesternUniversityFeinbergSchoolof
Medicine,Chicago,Illinois,UnitedStatesofAmerica,4TheFirstHospitalofWuhanCity,Wuhan,People’sRepublicofChina,5CenterforCardiovascularGenetics,
DepartmentofMolecularCardiology,LernerResearchInstitute,ClevelandClinic,Cleveland,Ohio,UnitedStatesofAmerica
Abstract
Background:
Peripheralarterialdisease(PAD)isacommondiseaseaccountingforabout12%oftheadultpopulation,and
euticangiogenesisusingangiogenicfactorshasbeenconsideredtobe
study,weassessedthepotentialofanewangiogenicfactorAGGF1for
therapeuticangiogenesisinacriticallimbischemiamodelinmiceforPAD.
MethodsandResults:
Wegeneratedaunilateralhindlimbischemiamodelinmicebyligationoftherightcommoniliac
icmicewithintrasmuscularadministrationofDNAforanexpressionplasmidforhuman
AGGF1(AGGF1group)resultedinincreasedexpressionofbothAGGF1mRNAandproteinaftertheadministrationcompared
withcontrolmicewithinjectionoftheemptyvector(controlgroup).ColorPWDopplerechocardiographyshowedthatthe
bloodflowinischemichindlimbswassignificantlyincreasedintheAGGF1groupcomparedtocontrolmiceattimepointsof
7,14,and28daysafterDNAadministration(n=9/group,P=0.049,0.001,and0.001,respectively).Increasedbloodflowin
theAGGF1groupwascorrelatedtoincreaseddensityofCD31-positivevesselsanddecreasednecrosisinmuscletissues
injectedwittoryimpairmentwas
significantlyreducedintheAGGF1groupcomparedtothecontrolgroup(P=0.004).TheeffectofAGGF1wasdose-
28aftergenetransfer,AGGF1wassignificantlybetterinincreasingbloodflowthanFGF-2(P=0.034),
althoughnodifferencewasfoundfortissuenecrosisandambulatoryimpairment.
Conclusions:
ThesedataestablishAGGF1asacandidatetherapeuticagentfortherapeuticangiogenesistotreatPAD.
Citation:LuQ,YaoY,YaoY,LiuS,HuangY,etal.(2012)AngiogenicFact
ONE7(10)::10.1371/.0046998
Editor:RajeshGopalraoKatare,UniversityofOtago,NewZealand
ReceivedFebruary20,2012;AcceptedSeptember11,2012;PublishedOctober23,2012
Copyright:ßanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisstudywassupportedbytheFundamentalResearchFundsfortheCentralUniversities(2010MS015,2011TS082),agrantfromtheStateKey
LaboratoryofFreshwaterEcologyandBiotechnology(2011FB16),aHubeiProvinceNaturalScienceKeyProgram(2008CDA047)andaKeyAcademicProgram
LeaderAwardofWuhanCity(2),andtheNationalNaturalScienceFoundationofChina(81070106).Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:qkwang@(QW);xtu@(XT)
Introduction
Peripheralarterialdisease(PAD)iscausedbyatherosclerosis,
whichresultsinprogressivenarrowingandocclusionofthe
peripheralarteriesandinhibitsbloodflowtothelowerextremities
[1,2].TheprevalenceofPADisincreasinginthemodernaging
society,andreachesabout12%oftheadultpopulation[3,4].In
theUS,approximately8to12millionpeopleareaffectedwith
PAD[5].InGermany,theprevalenceofPADforwomenand
menaged$65yearswas17%and20%,respectively[6].
TheclinicalpresentationofPADmayvaryfrombeing
asymptomatictopresentingwithaserioussymptomofintermittent
claudication[1,7].Inseverecases,PADsignificantlyaffectsquality
oflife,imately25%
patientswithcriticallimbischemiadieinoneyear[8].Moreover,
PLOSONE|1
inpatientswithPADtheprevalenceofcoronaryarterydisease
(CAD)isabout46%to71%[1,9,10],andatleast10%ofthem
sufferedcerebrovasculardisease[1,2,11,12].
ThecurrenttreatmentforPADfocusesondecreasing
cardiovascularandcerebrovascularmorbidityandmortalityand
onrelievingPADsymptoms[13].Pharmacologicaltreatments
withlipid-lowering,antihypertensive,andantithromboticmedica-
tionareusedtopreventmyocardialinfarctions(MIs)andstrokes
[1,13].SomePADpatientsmayrequirelegamputationtorelieve
d
amputationorotherseriousprognosis,angioplasty,stentingand
peripheralarterybypasssurgeriesareusedtorestorebloodflowto
thelegsinsomepatients[1,13,14].However,PADpatients
undergoingvascularsurgerieshadasignificantlyworselong-term
October2012|Volume7|Issue10|e46998
PotentialofAGGF1forTherapeuticAngiogenesis
sfuloverexpressionof
AGGF1
mRNAandproteininskeletalmusclebygenetransferusingaplasmid-baseddelivery
system.A)RealtimePCRanalysisforexpressionofhumanAGGF1mRNAderivedfromanAGGF1expressionplasmidinjectedintothegastrocnemius
-PCRprimersarespecifictohumanAGGF1andnotabletoamplifytheendogenousmouse
aare
shownastheamoudthecontaminationofplasmidDNA,RNA
tion,RT-PCRanalysiswithRNAsampleswithoutreversetranscriptiondidnotyieldanyproduct.
B)ExpressionofAGGF1proteiningastrocnemiusmusclesbyWesternblotanalysisattimepointsof7,14,28daysafterplasmidDNAwasinjected.
Theanti-AGGF1antibodyrecognizesbothhumanAGGF1derivedfromtheinjectedplasmidandendogenousmouseAGGF1protein.C)Theimages
fromWesternblotanalysisinB)werescanned,quantified,ensityofAGGF1signalwasnormalizedtothesignalofcontrolb-actin.
D)Immunohistochemicalanalysisofthesectionsofischemichindlimbmusclefrommiceinjectedwithanemptyvector(Control)andwithanAGGF1
expressionplasmid(AGGF1)withananti-AGGF1antibodysevendayslaterbyinjectionofplasmidDNA.*,P,0.05.
doi:10.1371/.0046998.g001
prognosisthanCADpatientswithsimilarprocedures[15].
Furthermore,manyPADpatientsarenotsuitablecandidatesfor
ore,therapeu-
ticangiogenesis,ionofangiogenesisandmicrocircula-
tionusingangiogenicfactors,hasbeenproposedasanewand
potentialtreatmentstrategyforPADpatientsinthelastdecade
[16].Theadministrationofangiogenicfactors,eitherasnaked
plasmidDNAorrecombinantproteins,maypromoteneovascu-
larization,augmentthecollateralcirculation,andenhanceblood
perfusiontoischemictissues[16,17].Therapeuticangiogenesisfor
PLOSONE|2
PADmadesomeprogress,butalsometsomeproblems[16,18].
Severalangiogenicfactors,forexample,vascularendothelial
growthfactorA(VEGF),fibroblastgrowthfactors(FGFs),
hepatocytegrowthfactor(HGF),andplatelet-derivedgrowth
factor(PDGF),havebeentestedtotreatcriticallimbischemiain
tudiesprovided
encouragingresults,however,therapeuticangiogenesisforPAD
isconsideredtobestillatitsinfancyandadverseeffectsinsome
casesoccurred,includingvascularleakage,transientedema,and
hypotensionwithadministrationofVEGFandFGFs[19,20].In
October2012|Volume7|Issue10|e46998
2024年4月6日发(作者：偶昭懿)
AngiogenicFactorAGGF1PromotesTherapeutic
AngiogenesisinaMouseLimbIschemiaModel
QiulunLu
1
,YihongYao
1
,YufengYao
1
,ShizhiLiu
1
,YuanHuang
1
,ShanLu
1
,YingBai
1
,BishengZhou
1
,
YanXu
1
,LeiLi
1
,NanWang
1
,LiWang
1
,JieZhang
1
,XiangCheng
2
,GangjianQin
3
,WeiMa
4
,ChengqiXu
1
,
XinTu
1
*,QingWang
1,5
*
1KeyLaboratoryofMolecularBiophysicsoftheMinistryofEducation,CollegeofLifeScienceandTechnology,CenterforHumanGenomeResearch,Cardio-XInstitute,
HuazhongUniversityofScienceandTechnology,Wuhan,People’sRepublicofChina,2InstituteofCardiology,UnionHospital,TongjiMedicalCollege,Huazhong
UniversityofScienceandTechnology,Wuhan,People’sRepublicofChina,3FeinbergCardiovascularResearchInstitute,NorthwesternUniversityFeinbergSchoolof
Medicine,Chicago,Illinois,UnitedStatesofAmerica,4TheFirstHospitalofWuhanCity,Wuhan,People’sRepublicofChina,5CenterforCardiovascularGenetics,
DepartmentofMolecularCardiology,LernerResearchInstitute,ClevelandClinic,Cleveland,Ohio,UnitedStatesofAmerica
Abstract
Background:
Peripheralarterialdisease(PAD)isacommondiseaseaccountingforabout12%oftheadultpopulation,and
euticangiogenesisusingangiogenicfactorshasbeenconsideredtobe
study,weassessedthepotentialofanewangiogenicfactorAGGF1for
therapeuticangiogenesisinacriticallimbischemiamodelinmiceforPAD.
MethodsandResults:
Wegeneratedaunilateralhindlimbischemiamodelinmicebyligationoftherightcommoniliac
icmicewithintrasmuscularadministrationofDNAforanexpressionplasmidforhuman
AGGF1(AGGF1group)resultedinincreasedexpressionofbothAGGF1mRNAandproteinaftertheadministrationcompared
withcontrolmicewithinjectionoftheemptyvector(controlgroup).ColorPWDopplerechocardiographyshowedthatthe
bloodflowinischemichindlimbswassignificantlyincreasedintheAGGF1groupcomparedtocontrolmiceattimepointsof
7,14,and28daysafterDNAadministration(n=9/group,P=0.049,0.001,and0.001,respectively).Increasedbloodflowin
theAGGF1groupwascorrelatedtoincreaseddensityofCD31-positivevesselsanddecreasednecrosisinmuscletissues
injectedwittoryimpairmentwas
significantlyreducedintheAGGF1groupcomparedtothecontrolgroup(P=0.004).TheeffectofAGGF1wasdose-
28aftergenetransfer,AGGF1wassignificantlybetterinincreasingbloodflowthanFGF-2(P=0.034),
althoughnodifferencewasfoundfortissuenecrosisandambulatoryimpairment.
Conclusions:
ThesedataestablishAGGF1asacandidatetherapeuticagentfortherapeuticangiogenesistotreatPAD.
Citation:LuQ,YaoY,YaoY,LiuS,HuangY,etal.(2012)AngiogenicFact
ONE7(10)::10.1371/.0046998
Editor:RajeshGopalraoKatare,UniversityofOtago,NewZealand
ReceivedFebruary20,2012;AcceptedSeptember11,2012;PublishedOctober23,2012
Copyright:ßanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted
use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisstudywassupportedbytheFundamentalResearchFundsfortheCentralUniversities(2010MS015,2011TS082),agrantfromtheStateKey
LaboratoryofFreshwaterEcologyandBiotechnology(2011FB16),aHubeiProvinceNaturalScienceKeyProgram(2008CDA047)andaKeyAcademicProgram
LeaderAwardofWuhanCity(2),andtheNationalNaturalScienceFoundationofChina(81070106).Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:qkwang@(QW);xtu@(XT)
Introduction
Peripheralarterialdisease(PAD)iscausedbyatherosclerosis,
whichresultsinprogressivenarrowingandocclusionofthe
peripheralarteriesandinhibitsbloodflowtothelowerextremities
[1,2].TheprevalenceofPADisincreasinginthemodernaging
society,andreachesabout12%oftheadultpopulation[3,4].In
theUS,approximately8to12millionpeopleareaffectedwith
PAD[5].InGermany,theprevalenceofPADforwomenand
menaged$65yearswas17%and20%,respectively[6].
TheclinicalpresentationofPADmayvaryfrombeing
asymptomatictopresentingwithaserioussymptomofintermittent
claudication[1,7].Inseverecases,PADsignificantlyaffectsquality
oflife,imately25%
patientswithcriticallimbischemiadieinoneyear[8].Moreover,
PLOSONE|1
inpatientswithPADtheprevalenceofcoronaryarterydisease
(CAD)isabout46%to71%[1,9,10],andatleast10%ofthem
sufferedcerebrovasculardisease[1,2,11,12].
ThecurrenttreatmentforPADfocusesondecreasing
cardiovascularandcerebrovascularmorbidityandmortalityand
onrelievingPADsymptoms[13].Pharmacologicaltreatments
withlipid-lowering,antihypertensive,andantithromboticmedica-
tionareusedtopreventmyocardialinfarctions(MIs)andstrokes
[1,13].SomePADpatientsmayrequirelegamputationtorelieve
d
amputationorotherseriousprognosis,angioplasty,stentingand
peripheralarterybypasssurgeriesareusedtorestorebloodflowto
thelegsinsomepatients[1,13,14].However,PADpatients
undergoingvascularsurgerieshadasignificantlyworselong-term
October2012|Volume7|Issue10|e46998
PotentialofAGGF1forTherapeuticAngiogenesis
sfuloverexpressionof
AGGF1
mRNAandproteininskeletalmusclebygenetransferusingaplasmid-baseddelivery
system.A)RealtimePCRanalysisforexpressionofhumanAGGF1mRNAderivedfromanAGGF1expressionplasmidinjectedintothegastrocnemius
-PCRprimersarespecifictohumanAGGF1andnotabletoamplifytheendogenousmouse
aare
shownastheamoudthecontaminationofplasmidDNA,RNA
tion,RT-PCRanalysiswithRNAsampleswithoutreversetranscriptiondidnotyieldanyproduct.
B)ExpressionofAGGF1proteiningastrocnemiusmusclesbyWesternblotanalysisattimepointsof7,14,28daysafterplasmidDNAwasinjected.
Theanti-AGGF1antibodyrecognizesbothhumanAGGF1derivedfromtheinjectedplasmidandendogenousmouseAGGF1protein.C)Theimages
fromWesternblotanalysisinB)werescanned,quantified,ensityofAGGF1signalwasnormalizedtothesignalofcontrolb-actin.
D)Immunohistochemicalanalysisofthesectionsofischemichindlimbmusclefrommiceinjectedwithanemptyvector(Control)andwithanAGGF1
expressionplasmid(AGGF1)withananti-AGGF1antibodysevendayslaterbyinjectionofplasmidDNA.*,P,0.05.
doi:10.1371/.0046998.g001
prognosisthanCADpatientswithsimilarprocedures[15].
Furthermore,manyPADpatientsarenotsuitablecandidatesfor
ore,therapeu-
ticangiogenesis,ionofangiogenesisandmicrocircula-
tionusingangiogenicfactors,hasbeenproposedasanewand
potentialtreatmentstrategyforPADpatientsinthelastdecade
[16].Theadministrationofangiogenicfactors,eitherasnaked
plasmidDNAorrecombinantproteins,maypromoteneovascu-
larization,augmentthecollateralcirculation,andenhanceblood
perfusiontoischemictissues[16,17].Therapeuticangiogenesisfor
PLOSONE|2
PADmadesomeprogress,butalsometsomeproblems[16,18].
Severalangiogenicfactors,forexample,vascularendothelial
growthfactorA(VEGF),fibroblastgrowthfactors(FGFs),
hepatocytegrowthfactor(HGF),andplatelet-derivedgrowth
factor(PDGF),havebeentestedtotreatcriticallimbischemiain
tudiesprovided
encouragingresults,however,therapeuticangiogenesisforPAD
isconsideredtobestillatitsinfancyandadverseeffectsinsome
casesoccurred,includingvascularleakage,transientedema,and
hypotensionwithadministrationofVEGFandFGFs[19,20].In
October2012|Volume7|Issue10|e46998