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Recombinant Human Platelet-derived Growth FactorEnhances Repa ir of Cutaneous Full-thicknessExcision by Increasing the Phosphorylationof Extracellular Signal-regulated Kinasein D iabetic RatCHEN G Biao1L I U Hongw ei1FU Xiaobing2SHEN G Zhiyong2SUN Tongzhu2【Abstract】ObjectiveTo investigate the possible signaling mechanism s by which recombinant human platelet2derived grow th factor(rhPDGF)accelerated healing of cutaneouswound in diabetic rats.M ethodsFour full2thicknessskin woundswere incised in the back of 26maleW istar diabetic rats.Thewounded ratswere divided into 3 groups(7 or8 rats each group).One group w ithout treatment was used as a control,and the other 2 groups were treated w ithrhPDGF at a dose of 710g?cm2wound or vehicle(DM SO?019%N aCl,vol?vol 11)from 1 to 14 days.The woundhealingwasevaluatedbythemeasurementsofthewoundvolumeandarea.I mmunofluorescentandi mmunohistochem ical staining were used to exam ine the phosphorylation of extracellular signal2regulated kinase 1?2(ERK1?2)and the expression of proliferative cell nuclear antigen(PCNA),respectively.ResultsGranulation tissueappeared in the bed of wound after injury.The number of blood capillary buds and fibroblasts was greater in therhPDGF2treated group than that in the other 2 groups.A lot of inflammatory cells infiltration and collagen depositionwere observed in the wound.The wound2volume in the rhPDGF2treated group was smaller than that in control group(P 0.05).The reepithelialization rate in rhPDGF2treated group was higher than that in the other 2 groups at 7 daysafter injury(P 0105).The expression of PCNA in reparative cellswas higher in rhPDGF2treated group than in controlgroup or vehicle2treated group at 3,7 days after injury(P 0105).The phosphorylation of ERK1?2 was stronger inrhPDGF2treated group than that in control group or vehicle group at 7 and 14 days after injury(P 0105).ConclusionThese results suggest that rhPDGF accelerates wound healing and i mproves healing quality by increasing thephosphorylation of ERK1?2.【Key words】D iabeticPlatelet2derived grow th factorWound healingExtracellular signal2regulatedkinaseFoundation item s:N ational Basic Science Research and Development Grants(2005CB522603);N ational N aturalScience Foundation of China(30170966,30230370);N ational Outstanding Young Researcher Foundation of China(39525024);N ational Postdoctoral Science Foundation of China(2001.14)BACKGROUNDT issuerepairinvolvesthecoordinatedinteraction of numerous cell types in the processes,includinginflammation,matrixdeposition,andremodeling,w hichrestorethecontinuityandarchitecture ofskin.The most commonchronicwoundsinclude pressureulcers,diabetic ulcers,arterial occlusive disease and venous ulcers.D iabetesmellitus is one1 Department of Plastic Surgery,Guangzhou General Hospital ofGuangzhou M ilitary Region,Guangzhou Guangdong,510010,P.R.China;2 Wound Healing and Cell Biology Laboratory,Institute forBasic Research,Trauma Center of Postgraduate M edical College,General Hospital of PLA,Beijing,100853,P.R.ChinaCorresponding author:FU Xiaobing,professor,Wound Healingand Cell Biology Laboratory,Institute for Basic Research,TraumaCenter of Postgraduate M edical College,General Hospital of PLA,Beijing,100853,P.R.China,E2mail:of the major contributors to chronic wound healingproblem s.D iabetic wounds represent a significanthealth care burden in the world1,2.The pathologicprocess of diabetici mpaired healingis complex.A lthough it is w ell accepted that diabetic woundshealpoorly,themechanism sunderlyingthisphenomenon are not totally understood.It has beendemonstrate that diabeticwounds have a deficiency ofgrow th factors,a prolonged inflammatory state,andi mpaired cellm igration and wound contraction3.Platelet2derived grow thfactor(PDGF)is apotentactivatorforcells4,w hichsti mulateschemotaxis,proliferation and new gene expression inmonocytes2macrophages and fibroblasts considered asessential celltypesfortissuerepair.PDGFisi mplicatedinmultipleaspectsofcellgrow th,differentiation,andsurvival viareceptor tyrosinekinases.PDGF could also directly induce endothelialcell proliferation and cord?tube formation during3901中国修复重建外科杂志2006年第20卷第11期angiogenesisin vitrovia PDGF B2receptors.RecentstudiesshowthatthePDGFisnecessaryforgranulation tissue formation and vascularization.Extracellularsignal2regulatedkinase1?2(ERK1?2),the mosti mportantcomponentofactivatedm itogen2activatedproteinkinases(MA PKs)fam ily is believed to translocate to thenucleus and regulate the expression of so2called earlygenes such as c2fos,c2myc and c2jun,w hich leads tow aves of more gene expression and ulti mately to acompletion of a specific cellular response such as cellm igration,proliferation and inflammation in somecells.It has been documentedthat ERK1?2 areactivated by grow th factor such as PDGF in the cellsassociated w ith wound healing.ToexploretheinvolvementofERKsinaccelerated wound healing by rhPDGF,w e developeda diabetic ratmodelof dermal full2thickness excision.W e compared the wound volume and area w ith orw ithouttreatmentofrhPDGF,exam inedthephosphorylation of ERKs in the wound of diabeticrats.MATERIALS AND M ETHODSAn i mals and surgical woundsTw enty2six adultmaleW istar diabetic rats(1602190 g)w ere used in this study.They w ere boughtfromChineseA cademyofM edicalSciences(certification:SCXK11000006).A ni mals w erehousedindividually andkeptina hum idity andtemperature controlled room under a 12212 h lightcycle(w ith lights on at 6 AM).Blood glucose levelsw ere measured.U nderpentobarbitalsodiumandketam ineanesthesia,hairs onthedorsumof m ice w ereclipped,and four full2thickness round skin wounds of2154 cm2(1.8 cm in diameter)w ere prepared usingskin punch equipment in the back of each rat.Thedistance betw een woundsw as at least 1.5 cm.Fifty2two wounds w ere divided into 3 groups:the woundw as not treated as a control(control group);thewounds w ere treated w ith rhPDGF2BB gel(rhPDGF2treated group)at a dose of 7.0g?cm2wound(REGRAN EX,OM J Pharmaceuticals,U SA)andw ith vehicle(DM SO?019%N aCl,vol?vol 11,vehicle2treated group),respectively.rhPDGF2BB gelw as topically applied to the wounds from 1 to 14days.V echicle2treated ani mals received equal amountof DM SO under si m ilar conditions.The rats w ereanesthetized w ith 10%chloral hydrate(350mg?kg),and killed after 3,7 and 14 days of operation.Twofull2thickness longitudinal incisions(2.0 cm)w eredone.Samplesw ere fixed in 10%buffered formalin,and embedded in paraffin and sectioned in 5.0m.Analysis of volume and reepithelializationinthewounded sitesGranulation tissue volume w as measured w ithinjectingnormalsalineinwoundsites,andreepithelialization w as calculated as a percent area ofthe originalwound size by using HE staining.Determ ination of collagen depositionThe presence of collagen fibers w as determ inedby light m icroscope in histologicalpreparations of theskin wound stained w ith V an Giesons at 7 days.Collagen fiber staining w as performed according to amethod described previously.The area of collagenper field in 3 groups w as exam ined by an i magingsystem consisting of a computer and m icroscope.I mmunohistochem ical sta in ingSpeci mens w erefixedin10%formalinandembedded in paraffin by routine procedures.Five2msections w ere cut fori mmunohistochem ical studyusingthelabeledstreptavidin2biotin2peroxidasemethodasdescribedpreviously.Thepri maryantibody used in this study w as anti2PCNA antibody(Santa CruzBiotechnology,Santa Cruz,CA,U SA).Briefly,endogenous peroxidase w as blocked w ith 0.3%hydrogen peroxidein methanol.Nonspecificbinding of the antibody w as blocked by 5%serum.Pri mary antibody diluted in 1%BA S in PBS w asapplied to the sections and incubated for 16224 hoursat4.Subsequently,biotinylatedsecondaryantibody and then streptavidin2peroxidase conjugatew ere applied.Positive staining w as visualized usingdiam inobenzidine,and nuclei w ere counterstainedw ith hematoxylin.N egative controls using normalmouseseruminsteadoftheindividual pri maryantiseraw erestainedbythesameprocedure.Sections w ere placed on the stage of the m icroscopew ithcharge2coupleddevicecamera(M itsubishi,Japan).Thenumber of proliferatingcells w asdeterm inedby manuallyscoringthenumber ofPCNA2positive staining cells at200 magnification.The PCNAlabelingindex,theratio of PCNA2positive cells vs.total cells,w as calculated.A totalof 6 random fields from either the leading woundmargin or the wound center(for closed wounds)w ere counted in each section by a blinded observer.The average index of three sections w as taken as thevalue for each wound.I mmunofluorescent sta in ingFormalin2fixedstripsw ereembeddedin4901Chinese Journal of Reparative and Reconstructive Surgery,2006,Vol 20(11)paraffin,and serial longitudinal 5m thick sectionsw ere cut.Every 10th section w as performed at roomtemperature.Non2specifici mmunoreactions w ereblocked by incubation for 20 m in w ith 10%goatserum in PBS.Sections w ere then incubated for 2 hw ith 1100 anti2p2ERK,w ashed 3 ti mes for 5 m inw ith PBS,and then incubated for 1 h w ith 11 000biotinylatedanti2mousei mmunoglobulinandstreptavidin2conjugatedTexasred.Sections w ereexam ined w ith O lympus m icroscope equipped w ith acomputer2basedi magingsystemandmeasuredfluorescenceintensityasevaluationofERKphosphorylation.Quantitative studyHE2stained wound sections w ere analyzed at40 magnification.Six sample areas per section of thewoundsin3groupsw ereexam inedbylightm icroscope.M orphologicalfindings,includingepithelialization,cellularcontent(neutrophils,macrophages,andfibroblasts),collagenregeneration,andvascularizationw erescoredaccording to a previously published system.Theresults w erejudged as follow s.Pale2yellow w asnegative(-),shallowbrow n w as w eak positive(+),brow n w asmoderately positive(),and deepbrow n w as strongly positive().Statistical analysisI mage collection w as performed by N IH i magesoftw are.The results w ere expressed as meansstandard deviation(SD).The data w ere analyzed byStudentspairedttest.Thedifferencew asconsidered to be significant ifP 0.05.RESULTSBasic glucose levels of diabetic ratsA ll diabetic rats used in the present study w eremarkedly hyperglycem ic w ith average glucose levelsof 233154 mmol?L,and show ed typical signs ofdiabetesconsistentw ithmodeling,includingpolydipsia,polyuria,and glycosuria.Effect of rhPDGF on reepithelialization of the woundThe effect of rhPGDF on reepithelialization w asdeterm ined by percentage of new epithelium in thetotal wound.TherateofreepithelializationinrhPDGF2treated group(4611%)w as higher than thatin vehicle2treated group(2312%)and control group(2516%)at 7 days after injury,show ing significantdifference(P 0.05,Fig.1 a,b).But the rate ofreepithelializationshow ed nosignificant differencebetw een rhPDGF2treated group and vehicle2treatedor control group at 14 day after injury.Effect ofrhPDGFonbloodvessel density andgranulation tissue arearhPDGF gel at a dose of 710g?cm2woundmarkedly increased blood vessel density and enhancedgranulation tissue formation at 7 days after injury.Effect of rhPDGF on healing qualif icationSignificanti mprovementinwoundhealinghistologyandwoundclosurew asfoundaftertreatment w ith rhPDGF gel at a dose of 7.0g?cm2wound.Typical adm inistration of rhPDGF promotedthe rate of wound closure and reepithelialization.rhPDGF enhanced formation of the granulation tissueand sti mulated neovascularization at 7 days afterinjury,the m icrovessel density in rhPDGF2treatedwounds w as higher than that in control or vehicle2treated wounds,and the diabetic rats treated w ithrhPDGFshow edagreaterhealingresponse.Grossly,rhPDGF2treated wounds became hyperem icat 3 days,and a rich,red granulation tissue bed w aspresent at 7 days.The wounds began to visuallyclose at 7 days,achieved complete reepithelializationat 14 days.A copious amount of edema fluid w asregularly noted in the rhPDGF2treated wound bedsfrom the 3rd to 7th day after injury,consistent w iththe permeability2inducingeffects ofrhPDGFonendothelium.D ifferences in mean wound sizes w ereobserved betw een different groups after 7 days.H istologically,more rapid epithelialization of thewound w as observed inrhPDGF2treated wounds.Granulation tissue deposition w as more abundant inrhPDGF2treated wounds at 7 and 14 days after inury(Fig.1c,d).Effect of rhPDGF on the expression of PCNAThe expression of PCNA w as distinguishable at3 days after injury in diabetic rats.PCNA proteinlevels show ed a marked up2regulation in control andvehicle2treated groupat 7 days after injury,theexpression of PCNAprotein remained roughly atthese levels throughout the rest of the observationperiod.A dm inistrationofexogenousrhPDGFresulted in the significant increase in the expressionofPCNAinthe wound ofdiabeticrats w hencompared w ith control or vehicle2treated group at 3and 7 days after injury(Tab.1),as show n in Fig.2.Effect of rhPDGF on the phosphorylation of ERK1?2A s show n in Fig.3 a,b,the phosphorylation ofERK1?2 positive cells w as distinguishable at 3 daysafterinjuryindiatbeticrats.A fter 7 daysthepositive cells of ERK1?2 phosphorylation w as furtherincreased in the wound of diabetic rats.Follow ing5901中国修复重建外科杂志2006年第20卷第11期thispeak,therestillw asexpressionofphosphorylation of ERK1?2 at 7 and 14 days afterinjury.The adm inistration ofrhPDGF markedlyincreased ERK 1?2 phosphorylation in the wounds ofdiabetic rats at 7 and 14 days after injury(P 0105),as show n in Tab.2 and injury Fig.3 c,d.Tab.1Effect of rhPDGF on the expression of PCNA in diabetic ratsGroupTi me after injury(d)3714Control group204386334Vehicle2treated group225407325rhPDGF2treated group356363733743Compared w ith control group or vehicle2treated group,P 0105Tab.2Effect of rhPDGF on the phosphorylation of ERK1?2 indiabetic ratsGroupTi me after injury(d)3714Control Group1 0842102 4802251 340170Vehicle2treated group1 1402402 6302501 510190rhPDGF2treated group1 3902503 98031032 28025033Compared w ith control group or vehicle2treated group,P 0105D ISCUSSI ONM any grow th factors play a crucial role in theinitial phases of wound healing.Grow th factors mayexert their effect on target cells in either a paracrine,autocrine,inter2crine(acting w ithin the cell thatcreated them),or endocrine fashion.A lmost all arepeptides that bind to the target cell by w ay of high2affinity cell surface receptor proteins.The receptorbinding causes intracellular reactions that are notentirely understood.PDGF is released from plateletsshortly after a thrombus is formed and hemostasis isachieved.The events of early acute wound healingreflectafinelybalancedenvironmentinw hichproteolytic activity and matrix synthesis occur undertight regulation,leading to uncomplicated and rapidwound healing.Chronic wounds,for one reason oranother,have lost this fine balance.U nderstandingthe alteration of the grow th factor equilibriuminchronic wounds may allowfor the better use ofgrow th factors toassistinthe healing oftheseproblematic wounds.Chronic,no2healing woundsare characterize
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