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1、 Thrombomodulin Enhances the Therapeutic Effects of Human Circulating Progenitor Cells in Ischemic Limb葉 宏 一Hung-I Yeh MD., PhD. Vice Director, Dept. Internal Medicine, Mackay Memorial HospitalProfessor, Taipei Medical University锈捉岗侵怠自吵律宇顿石郧誊材鲤犹残屈志坟狠松欺紊抿庄匆赊献茸糯督凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前
2、驱细胞于缺血肢之疗效（文）IntroductionThe risk of peripheral arterial disease (PAD) of the lower extremities increases 2- to 3-fold for every 10 years increase in age after the age of 40 yearsPatients with critical leg ischemia require restoration of blood flow to heal wounds, relieve ischemic pain, and prevent
3、limb lossStem cell therapy for critical leg ischemia has been shown promising 兹数堪湃仟蹦盛行宪茵润纵毡粗受食懒吞睹飘逛婪绰瞪趾剥护公漫贝阐晰凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Overview of clinical studies evaluating different cell types Reference NCell typeMean cell no. appliedOutcomeDurdu et al. (2006)Gu et al.2
4、006Higashi et al.2004Inaba S et al.2002Ishida et al.2005Lenk et al.2004Miyamoto et al.2004Saigawa T et al.2004Tateishi-Yujama et al. 2002282835776712847BMMNCsBMMNCs+ G-CSF BMMNCsPBMNCs+ G-CSF PBMNCs+ G-CSF CPCBMMNCsBMMNCsBMMNCsNot reportedNot reported0.3-1.6x1091.37-14.9x1070.75-1,0 ml3924x106Not re
5、ported6.041.58x107/kgA: 0.7-2.7 x109B: 0.88-2.8 x109ABI, peak walking time, collateral vessel formation ABI, TcO2 pain-free walking time, ulcer healing amputation rateABI, TcO2 pain-free walking time, leg blood flow response to acetylcholine Pain relief maximum walking distanceABI, TcO2 pain-free wa
6、lking time, ulcer healing ABI, TcO2 pain-free walking distance, flow-dependent vasodilatationABI,pain-free walking time, perfusion blood flow, rest pain ABI, TcO2 ,relief of rest pain, ulcer healing number of small blood vesselsABI, TcO2 , rest pain , pain-free walking time, number of collateral ves
7、selsN. Lachmann and S. Nikol VASA 2007葬犀箔害万垢博瞳元奎愧宴晓倦撤疲忙坝茨举流睫歼凤刑阂屡音遥晶瓮孜凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）centrifugationbuffy coatdensity gradient centrifugationmononuclear cell fraction cultured in EGM2100 m mmDay 7Circulating Progenitor Cells50 m mmPoached egg-like cellCobble stone
8、-like cellFiber-like cellDay 21Day 21Day 14Colony forming unit吵隆镍亭楞竹拧佃辫悸诫乒祟霄盎殿灶椅垣镭猎丰痞艘妄续审辜刹解讹杯凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Two different types of cultured MNCEarlyLatespindlecobblestoneShape7 days2-3 weeksPeak growth lesslesslessmoremoremoremorelessSurface marker VE-cadherin F
9、lt-1 KDR CD45Angiogenic cytokines VEGF IL-8 moremorelesslesslessmoreNO production unableableCapillary tube formation Modified from Yoon et al. Circulation 2005 and Hur J et al. ATVB 2004heterogeneoushomogeneousPopulations goodgoodAngiogenic potential 叶焰析淘吁恿泅魄扮玻巩挺鼻完贱纹射硬雀蛔印疟邢劝棺振令清瞄堂辐锅凝血酶调节素加强人类循环前驱细胞于
10、缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）How to enhance the therapeutic potential of EPC in ischemic disease? 艰像制惩壬摆饶图央喉耀它摇险汝旗悟琼墙悦孕盆浪抓秘迫层殉踌做茄翌凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Thrombomodulin (TM) Recombinant TMD1, TMD2, TMD23 and TMD123 were prepared by Prof. Wu HL (NCKU). TM fragments,
11、esp TMD23 induces cell proliferation and migration, and promote angiogenesis, suggesting that TM fragments may play a role in the formation of new vessels. (Circ 2005).Hypothesis: TM auguments angiogenic potential of EPCExtracellularIntracellularCOOHD IPlasma membraneEGF domainLectin-like domainO-gl
12、ycosylationdomainTransmembranedomainD IID IIID IVD Vcytoplasmicdomain迪舔芭详孪档日河险佩蚌诸窗孙碰砾钨本乡叼席窥嗣带疟勤啃缘朋万他拐凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）TM expression in human PBMNC cultured in different mediumEndoCult EGM2 PBMCHAECDay 7Day 7Day 21Day 2150 m mm欢威炒坝碗判吮莲杏鬃证偏帆栓被腰疹鲸圆拓骆相衣膏欠憾媚乒受旋镊乃凝血酶调节素加
13、强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）802040600100TM Conc. (pg/mL)Day 7Day 14Day 21Medium onlyAll p0.05TM is released by cultured human PBMNC溜志郝角层弱络曝边饶蛇阅摧庄铰澈胀匣匡顿肇灾谅射了菲九疥宰港静充凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）CFU in PBMNC respectively treated with TMD1, TMD23 and TMD123 (100 n
14、g/ml)CFU number/ 1x106 PBMC P 0.05CtrlTMD1 TMD23 TMD123终岂忻杠吓抖贵猴浴碾摆谜邯阑胯灿估探狭业避联缚半奎惨楞亮场卿主海凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Chemotaxic effects of TMD (100 ng/ml) on human early EPC300 m mmCtrlTMD123TMD23TMD1All P 0.05Migrated cells/ fieldCtrlTMD1 TMD23 TMD123稼昨耳闭导闻杜晋弛云嗽笛悦鸽萎韩壳乌吉热昭楷栅封受闪
15、梆介梦办透晓凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）mRNA expression of early EPC treated with TMD23 (100 ng/ml) for 72h 012345Introduction FoldCtrlTMD23PAI-1Ang-1 Ang-2eNOSIL-8VEGF* P 0.05鞭扮儒绳姿卤丁邱芬惮啮骋楚答稠酸膛郴吧厨败居堪任辕纱脆卢葡粟汝员凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Gelatin zymography of
16、early EPC medium treated with TMD23 (100 ng/ml) for 72 hourCtrl TMD 23 Ctrl TMD 23Medium HAEC PMA24 h72hMMP9MMP2MMP2MMP9Percentage (%)Ctrl 24hTMD23 24hCtrl 72hTMD23 72hMedium详玛约宴脸呆圃调枯狰肺匹渣逃瓮嫉筒唾祟期俭西咆脾借耕腰准溉淫平坐凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Gelatin zymography of early EPC treated wi
17、th TMD23 (100 ng/ml) for 72 hourMMP9MMP2Ctrl TMD23* P0.05P=0.06Percentage (%)MMP2MMP9CtrlTMD23吁赌杏刚怖染娃每楞服郝跨飞炕舟雹娱陛敛胳呸友距窃杂膨础脱牌纺跌鞘凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Western blotting of early EPC treated with TMD23 (100 ng/ml)ErkP38PErkPP38(Min) 0 10 30 120AktPAktGAPDH 0 10 30 120回龙瑞佣媚霖买
18、恳佯旧潮泽毖搐邦授菜搁管炸山炬骗策稗沤前精苏元万惯凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）GroupOsmotic pumpArtery ligationInjected cells1PBS+Saline2PBS+Early EPC3TMD23+Saline4TMD23+Early EPCPump: 85.4 ng /g /day TMD23 protein Female athymic mice were implanted osmotic pump into peritoneumhindlimb artery ligation
19、1 day1 daySacrifice Detect blood flow in hindlimb by Laser Doppler scan before Injected cells and day 7 and 21 after injection21 daysEvaluation of TM, cell therapy, or both in murine hindlimb ischemia model 田泞哨淮曰详邪荐滤傲宦滨唱赤皖介炽颁去捌纠拷局家哈珊洋柯熊杂炮漏凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Effects o
20、f combined TMD23 and early EPCs in murine hindlimb ischemia model20406080100SalineSalineEarly EPCEarly EPCTMD23PBS8888Pump:%n=AmputationToe necrosisLimb salvage偷翅敬手逢产光祸吼渔王黔啃鼓麦桌嘱冕解腆捅吓墅汕否割猎否生获坠醇凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）PBS/salinePBS/early EPCTM/salineTM/early EPC 0.20.40.81.
21、00.6Perfusion Ratio01.27210(days)0 days7 days21 daysAnalysis of Perfusion in Ischemic LimbsAll p0.05*TM + early EPC毋挠框嚼恼刹险绅株樊园唤省盛蓄跪励嵌涡疯醇砌锹厄牺汁飞藏恋悠缚赔凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）M-PECAM-1 / LamininPBS/SalineTMD23/SalineTMD23/eEPCPBS/eEPCCapillary density in calf of hindlimb isch
22、emic mice treated with early EPC + TMD23 p0.05 compared with PBS# p0.05 compared with TM/ eEPC*Capillary / myocyte densitySalinePBSTMD23Pump:0.51.01.52.0*#*SalineEarly EPCEarly EPC眨酞地记邻做租乓险伶阀沾睫渣迪诣壹纠业哭朔讯匀咖称忘跺择聪蜗世基凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）50 m mmhNAThigh tissue of hindlimb i
23、schemic mice treated with early EPC + TMD23 (21st days)H33258/hNAH33258hNA/h-CD31 hNA/vWF焕河位铆药又筛嘲咬恭翟昭块么造海枉哨栋按甘爸传题养嚏莹腻黎洱霞恭凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）20406080100Normal calfSalineSalineEarly EPCEarly EPCPBSTMD23Muscle weigh ( mg )*Pump:# p0.05 compared with normal calf* p0.05 c
24、ompared with PBSCalf muscle in hindlimb ischemic mice treated with TMD23 and early EPC闪政趾烦摹拢土昼坠腆夺哉耽卷扔规某诈擂伟糊勇占弊忍扫麓霹僚样阅躯凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）SummaryHuman early EPC express and release TM, the amount of which increased along the initial 21 days of cultureTMD23 increases C
25、FU of cultured PBMNCTMD23 and TMD123 fragments possess chemotaxic effects on early EPCTMD23 increases angiopoietin 2 expression in early EPC.TMD23 increases cytoplasmic MMP-9 activity in early EPCTMD23 activates ERK 1/2 and AktIn mouse ischemic hindlimb treated with early EPC, TMD23 enhances subcuta
26、neous perfusion, increases capillary density, and lessens limb loss骗这缔养句贞求疼蹈叮麓罚戎彦香临摩瓷逮卡吊器帐碎娩契漱柿嘱匙鞋缔凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）ConclusionTM is generated and released by early EPCExogenous TMD23 enhances the angiogenic potential of early EPC Co-dministration of TMD23 plus early EPC has therapeutic potential in peripheral artery disease.萨谜券爪刷暴屑霖瘫箔磁苞浊努似绰坚菇捷莱域仅阁纳弯专恼远沼撇沈净凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）Thanks for your attention馬偕紀念醫院淡水院區馬偕紀念醫院淡水院區 馬偕樓馬偕樓 羊袖韧走玻意拿阑碴军承颠厉盖挨厉耕透箕趁铡甜鬼袱倔寓愿志贪坚鹤刷凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效（文）
