EXPRESSION AND ROLE OF PLASMINOGEN SYSTEM IN PROCESS OF RESTENOSIS

Journal of Shanghai Second Medical University 2005 Vol.17 No. 2EXPRESSION AND ROLE OF PLASMINOGEN SYSTEM INPROCESS OF RESTENOSISZHAO Hai-guang(赵海光)，LU Xin-wu(陆信武)，HUANG Ying( 黄英)，JIANG Mi-er( 蒋米尔)Department of Vascular Surgery , Ninth People' s Hospital , Shanghai Second Medical University , Shanghai 200011 ,ChinaAbstractObjective To study the expression and role of plasminogen system in the process of restenosis.Methods We established a double-injury model of atherosclerotic restenosis in rabbit iliac artery mimicking humanarterial restenosis. The time course of tissue plaminogen activator( tPA ) , urokinase plasminogen activator( uPA ) ,urokinase plasminogen activator receptor ( uPAR ) and plasminogen activator inhibitor-I ( PAI-I ) was investigatedby immunohistochemistry. The mRNA expression of uPA and uPAR were detected after vascular procedures by in situhybridization. Results In uninjured arteries , the weak expression of tPA and PAI-I was detected in intimal andendothelial cells. The expression of tPA , uPA , uPAR and PAI-I was significantly induced afier double-injury , butafier double -injury 14d , the expression of tPA restore to preinjury levels. The expression of uPA and uPAR in intimalwas higher than that of media and maintain high levels in intimal within 42d and 56d.Conclusion Whereast-PA is primarily involved in clot dissolution and play a linited role in the process of restenosis , in plasminogen sys-tem , uPA and uPAR play a prominent role in the process of restenosis.Key wordsplasminogen systemexpressionrestenosisThe plasminogen system is made up of plasmino-the second operation ( 5 rabbits per team ). Eachgen , plasminogen activator and its receptor , plasminfemoral artery segment was postfixed as requested.and plasminogen activitor inhibitor. Many researchImmunohistochemistryImmunohistochemis-has proved that the expression of plasminogen systemtry was used to detect the expression of the plasmino-was obviously increased after the reconstructive vascu-gen activator( uPA )and its receptor( uPAR ) , tissuelar operation. But the role of the component of theplasminogen activator( tPA )and plasminogen activatorplasminogen system during this process of restenosis isinhibitor-1( PAI-1 ). tPA antiboday( 200μg/mL ),still unclear! 2]. The present research was done to re-uPA antibody( 100μg/ mL，rabbit antiserum to hu-search its expression and role during the process ofman ) , uPAR antibody( 100μg/ 'mL , rabbit antiserumrestenosis after the reconstructive vascular operation.to human ) ,PAI-1 antibody( 50μg/ mL , rabbit antise-rum to human )second antibody( 200μg/ mL ,mouseMATERIALS AND METHODSantiserum to rabbit )were all purchased from the Jing-mei Company. The procedure was : put the slidesAnimalsThirty-five New Zealand rabbits ,in the ethanol( 100% , 95% , 75% ) , then flushedweighing 2.5 -3. lkg , atherosclerosis was developedthem with tap-water for 2min ; dip them in the 1%in both iliac arteries as previously describedl. lcmpro中国煤化工the antigen ; soaked insegment was taken out of each experimental and con-thelded 0.3% H202 50μLYH.CNMHGtralateral iliac artery at the time of right after thefor sumn , ltusneu witn rD three times , 2min forsecond operation and3 ,7 ,14 ,28 ,42 and 56d aftereach time ; added 10% calf blood serum 50μL forSupported by grants. from National Natural Science Foundation of China( 30471688 ) , Shanghai Scientfic Committe( 0041 19022 ) , and ShanghaiMedical Burobu西数据6115 ).Journal of Shanghai Second Medical University 2005 Vol.17 No, 230min ; flushed with PBS three times , 2min for each37C , l5min x 1 ; added blocking solution ,37C ,time ;dilute the first antibody with PBS( tPA1 :50 ,30min ; added mouth anti-Dig 20μL , 37°C ,lh ;uPA1 10 ,uPAR1 :10 ,PAI-11 100 ); add the anti- .flushed with 0.5mol/L TBS 5min x4 ; added SABC-dilution of the first antibody 50μL ,put into the wet .AP 379C 20min ; flushed 0.5mol/L TBS 5min x4 ;box , then stayed overnight in 4C refrigerator ; tookadded BCIP/NBT 37C ,10min ; flushed with ddH20outthewetbox,flushitwithPBS3times,2minto terminate reaction ; block slides ; observated sam-each time ; added the second antibody 50μL 50minples with microscope and took pictures.under the room temperature ; flushed with PBS for 3tatistical analysis Data are expressed as x士s.times ,2min for each time ; added DAB 50μL forThe segments for immunohistochemistry and hybrid-each slide , terminate the reaction with flushing wa-ization in situ were analyzed by the computer imageter after 2min ; dip the slides in the Harris foranalysis systerm ( OLYMPUS image record systerm0.5min , then flushed them with water for 5min ;dipand PRO-IMAGE image treatment software ). Com-into 95% ,100% ethanol , each for 2min ; treatedpared the image of the experimental side segment andwith dimethylbenzene then blocked with neutral gum.the control side segment. Calculate the difference ofHybridization in situ Hybridization in situ kitthe grey level value of the intima and media of eachwas purchased from the Boshide Company( MK1436 ).side. Data of each side were analyzed by the StudentOligonucleotide probe of uPAR :5' -ACACT GCATGt test.CACTF CAGGC CCCAA GAGGC-3' , 5'-AGGCTRESULTSTTGCT GCCGG CCCCT CTCAC AGCTC3' ,5'-CCAGA CATTG ATTCA TTCCT CCTCG GCAGT-tPA expressed mildly in the endothelial cell and3'. Oligonucleotide probe of uPA : 5' -CCTGC AGthe intima of the normal blood vessel wall. 3d and 7dTAA TTGTG TTTCC CCAGG CCTAG-3' ,5'-TCCAGGAAGT GTGAG ACCCT CGTGT AGACA- 3'. Theafter the second injury , the expression of TPA of theexperimental side increased obviously , especially forprocedure was : took out iliac artery from both sides ,the media and intima. But there is no obvious differ-fixated with 4% PFA , stayed overnight under 4C ;ence between media and intima. After 14d , the ex-cleaned the samples with 0. 01mol/L PBS ; desicca-pression of TPA decreased obviously and returned toted the samples with 20% saccharu solution , stayedthe level before the second injury. The expression ofovermight under 4C ; embedded the samples withuPA and uPAR changed almost synchronously. TheyOCT，congulated under 一 209C ，preserved underwere not detected in the normal blood vessel wall. 3d- 70C ; cut into section , dehydrated under roomafter the second injury , they were detected in thetemperature ; flushed with 0. 0lmol/L PBS ,5min xblood vessel wall of the experimental side. The ex-2 ; tripsinized for lmin under 37C ; flushed withpression reached the climax on d14. On d28 , the ex-0.01mol/L PBS 5min x3 ; flushed with DEPC 5minpression of uPA and uPAR decreased , but still stayedx l ; forcehybridization : added forcehybridization so-中国煤化工! d56. During the earlylution 20μL for each slide ,379C ,4h ; hybridization :perYHCNMHGd3andd7),uPAandadded Dig probe 20μL( 1. 5μug/mL ) to each slide ,uPAR expressed in the full layer of blood vessel wall,stayed overnight under 37C ; flushed with 2 x SSCbut most obviously at the boundary of the media andunder 37C ,5min x2 ; flushed with 0.5 x SSC underintima. After d14 ，they expressed mostly in the37°C ,15品方效据flushed with 0. 2 x SSC undermedia and intima , and the expression in the intima isJournal of Shanghai Second Medical University 2005 Vol.17 No. 2much more obvious than that in the media ( P