凝血酶调节素加强人类循环前驱细胞于缺血肢之疗效(英文)课件

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 UniversityIntroductionThe risk of peripheral arterial disease (PAD) of the lower

2、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 limb lossStem cell therapy for critical leg ischemia has been shown promising Overvi

3、ew of clinical studies evaluating different cell types Reference NCell typeMean cell no. appliedOutcomeDurdu et al. (2006)Gu et al.2006Higashi 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-

4、CSF BMMNCsPBMNCs+ G-CSF PBMNCs+ G-CSF CPCBMMNCsBMMNCsBMMNCsNot reportedNot reported0.3-1.6x1091.37-14.9x1070.75-1,0 ml3924x106Not reported6.041.58x107/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 rateA

5、BI, TcO2 pain-free walking time, leg blood flow response to acetylcholine Pain relief maximum walking distanceABI, TcO2 pain-free walking time, ulcer healing ABI, TcO2 pain-free walking distance, flow-dependent vasodilatationABI,pain-free walking time, perfusion blood flow, rest pain ABI, TcO2 ,reli

6、ef of rest pain, ulcer healing number of small blood vesselsABI, TcO2 , rest pain , pain-free walking time, number of collateral vesselsN. Lachmann and S. Nikol VASA 2007centrifugationbuffy coatdensity gradient centrifugationmononuclear cell fraction cultured in EGM2100 mmDay 7Circulating Progenitor

7、 Cells50 mmPoached egg-like cellCobble stone-like cellFiber-like cellDay 21Day 21Day 14Colony forming unitTwo different types of cultured MNCEarlyLatespindlecobblestoneShape7 days2-3 weeksPeak growth lesslesslessmoremoremoremorelessSurface marker VE-cadherin Flt-1 KDR CD45Angiogenic cytokines VEGF I

8、L-8 moremorelesslesslessmoreNO production unableableCapillary tube formation Modified from Yoon et al. Circulation 2005 and Hur J et al. ATVB 2004heterogeneoushomogeneousPopulations goodgoodAngiogenic potential How to enhance the therapeutic potential of EPC in ischemic disease? Thrombomodulin (TM)

9、Recombinant TMD1, TMD2, TMD23 and TMD123 were prepared by Prof. Wu HL (NCKU). TM fragments, 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 pote

10、ntial of EPCExtracellularIntracellularCOOHD IPlasma membraneEGF domainLectin-like domainO-glycosylationdomainTransmembranedomainD IID IIID IVD VcytoplasmicdomainTM expression in human PBMNC cultured in different mediumEndoCult EGM2 PBMCHAECDay 7Day 7Day 21Day 2150 mm802040600100TM Conc. (pg/mL)Day 7

11、Day 14Day 21Medium onlyAll p0.05TM is released by cultured human PBMNCCFU in PBMNC respectively treated with TMD1, TMD23 and TMD123 (100 ng/ml)CFU number/ 1x106 PBMC P 0.05CtrlTMD1TMD23TMD123Chemotaxic effects of TMD (100 ng/ml) on human early EPC300 mmCtrlTMD123TMD23TMD1All P 0.05Migrated cells/ fi

12、eldCtrlTMD1TMD23TMD123mRNA expression of early EPC treated with TMD23 (100 ng/ml) for 72h 012345Introduction FoldCtrlTMD23PAI-1Ang-1Ang-2eNOSIL-8VEGF* P 0.05Gelatin zymography of early EPC medium treated with TMD23 (100 ng/ml) for 72 hourCtrl TMD 23 Ctrl TMD 23Medium HAEC PMA24 h72hMMP9MMP2MMP2MMP9P

13、ercentage (%)Ctrl 24hTMD23 24hCtrl 72hTMD23 72hMediumGelatin zymography of early EPC treated with TMD23 (100 ng/ml) for 72 hourMMP9MMP2Ctrl TMD23* P0.05P=0.06Percentage (%)MMP2MMP9CtrlTMD23Western blotting of early EPC treated with TMD23 (100 ng/ml)ErkP38PErkPP38(Min) 0 10 30 120AktPAktGAPDH 0 10 30

14、 120GroupOsmotic 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 ligation1 day1 daySacrifice Detect blood flow in hindlimb by Laser Doppler scan b

15、efore Injected cells and day 7 and 21 after injection21 daysEvaluation of TM, cell therapy, or both in murine hindlimb ischemia model Effects of combined TMD23 and early EPCs in murine hindlimb ischemia model20406080100SalineSalineEarly EPCEarly EPCTMD23PBS8888Pump:%n=AmputationToe necrosisLimb salv

16、agePBS/salinePBS/early EPCTM/salineTM/early EPC 0.20.40.81.00.6Perfusion Ratio01.27210(days)0 days7 days21 daysAnalysis of Perfusion in Ischemic LimbsAll p0.05*TM + early EPCM-PECAM-1 / LamininPBS/SalineTMD23/SalineTMD23/eEPCPBS/eEPCCapillary density in calf of hindlimb ischemic mice treated with ea

17、rly EPC + TMD23 p0.05 compared with PBS# p0.05 compared with TM/ eEPC*Capillary / myocyte densitySalinePBSTMD23Pump:0.51.01.52.0*#*SalineEarly EPCEarly EPC50 mmhNAThigh tissue of hindlimb ischemic mice treated with early EPC + TMD23 (21st days)H33258/hNAH33258hNA/h-CD31 hNA/vWF20406080100Normal calf

18、SalineSalineEarly EPCEarly EPCPBSTMD23Muscle weigh ( mg )*Pump:# p0.05 compared with normal calf* p0.05 compared with PBSCalf muscle in hindlimb ischemic mice treated with TMD23 and early EPCSummaryHuman early EPC express and release TM, the amount of which increased along the initial 21 days of cultureTMD23 increases CFU of culture