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 Hospital Professor, Taipei Medical University,Introduction,The risk of peripheral arterial disease (PAD) of the lo
2、wer extremities increases 2- to 3-fold for every 10 years increase in age after the age of 40 years Patients with critical leg ischemia require restoration of blood flow to heal wounds, relieve ischemic pain, and prevent limb loss Stem cell therapy for critical leg ischemia has been shown promising,
3、Overview of clinical studies evaluating different cell types,Reference,N,Cell type,Mean cell no. applied,Outcome,Durdu et al. (2006),Gu et al. 2006,Higashi et al. 2004,Inaba S et al. 2002,Ishida et al. 2005,Lenk et al. 2004,Miyamoto et al. 2004,Saigawa T et al. 2004,Tateishi-Yujama et al. 2002,28,28
4、,35,7,7,6,7,12,8,47,BMMNCs,BMMNCs + G-CSF,BMMNCs,PBMNCs + G-CSF,PBMNCs + G-CSF,CPC,BMMNCs,BMMNCs,BMMNCs,Not reported,Not reported,0.3-1.6x109,1.37-14.9x107,0.75-1,0 ml,3924x106,Not reported,6.041.58x107/kg,A: 0.7-2.7 x109 B: 0.88-2.8 x109,ABI, peak walking time, collateral vessel formation ,ABI, TcO
5、2 pain-free walking time, ulcer healing amputation rate,ABI, TcO2 pain-free walking time, leg blood flow response to acetylcholine ,Pain relief maximum walking distance,ABI, TcO2 pain-free walking time, ulcer healing ,ABI, TcO2 pain-free walking distance, flow-dependent vasodilatation,ABI,pain-free
6、walking time, perfusion blood flow, rest pain ,ABI, TcO2 ,relief of rest pain, ulcer healing number of small blood vessels,ABI, TcO2 , rest pain , pain-free walking time, number of collateral vessels,N. Lachmann and S. Nikol VASA 2007,centrifugation,buffy coat,density gradient centrifugation,mononuc
7、lear cell fraction,cultured in EGM2,100 mm,Day 7,Circulating Progenitor Cells,50 mm,Poached egg-like cell,Cobble stone-like cell,Fiber-like cell,Day 21,Day 21,Day 14,Colony forming unit,Two different types of cultured MNC,Early,Late,spindle,cobblestone,Shape,7 days,2-3 weeks,Peak growth,less less le
8、ss more,more more more less,Surface markerVE-cadherinFlt-1KDRCD45,Angiogenic cytokinesVEGFIL-8,more more,less less,less,more,NO production,unable,able,Capillary tube formation,Modified from Yoon et al. Circulation 2005 and Hur J et al. ATVB 2004,heterogeneous,homogeneous,Populations,good,good,Angiog
9、enic potential,How to enhance the therapeutic potential of EPC in ischemic disease?,Thrombomodulin (TM),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 fragmen
10、ts may play a role in the formation of new vessels. (Circ 2005).,Hypothesis: TM auguments angiogenic potential of EPC,Extracellular,Intracellular,COOH,D I,Plasma membrane,EGF domain,Lectin-like domain,O-glycosylation domain,Transmembrane domain,D II,D III,D IV,D V,cytoplasmic domain,TM expression in
11、 human PBMNC cultured in different medium,EndoCult,EGM2,PBMC,HAEC,Day 7,Day 7,Day 21,Day 21,50 mm,TM is released by cultured human PBMNC,CFU in PBMNC respectively treated with TMD1, TMD23 and TMD123 (100 ng/ml),CFU number/ 1x106 PBMC,P 0.05,Ctrl,TMD1,TMD23,TMD123,Chemotaxic effects of TMD (100 ng/ml
12、) on human early EPC,300 mm,Ctrl,TMD123,TMD23,TMD1,All P 0.05,Migrated cells/ field,Ctrl,TMD1,TMD23,TMD123,mRNA expression of early EPC treated with TMD23 (100 ng/ml) for 72h,0,1,2,3,4,5,Introduction Fold,Ctrl,TMD23,PAI-1,Ang-1,Ang-2,eNOS,IL-8,VEGF,* P 0.05,Gelatin zymography of early EPC medium tre
13、ated with TMD23 (100 ng/ml) for 72 hour,Ctrl TMD23,Ctrl TMD23,Medium HAECPMA,24 h,72h,MMP9,MMP2,MMP2,MMP9,Percentage (%),Ctrl 24h,TMD23 24h,Ctrl 72h,TMD23 72h,Medium,Gelatin zymography of early EPC treated with TMD23 (100 ng/ml) for 72 hour,MMP9,MMP2,Ctrl TMD23,* P0.05,P=0.06,Percentage (%),MMP2,MMP
14、9,Ctrl,TMD23,Western blotting of early EPC treated with TMD23 (100 ng/ml),Erk,P38,PErk,PP38,(Min) 0 10 30 120,Akt,PAkt,GAPDH,0 10 30 120,Pump: 85.4 ng /g /day TMD23 protein,Female athymic mice were implanted osmotic pump into peritoneum,hindlimb artery ligation,1 day,1 day,Sacrifice,Detect blood flo
15、w in hindlimb by Laser Doppler scan before Injected cells and day 7 and 21 after injection,21 days,Evaluation of TM, cell therapy, or both in murine hindlimb ischemia model,Effects of combined TMD23 and early EPCs in murine hindlimb ischemia model,20,40,60,80,100,Saline,Saline,Early EPC,Early EPC,TM
16、D23,PBS,8,8,8,8,Pump:,%,n=,PBS/saline,PBS/early EPC,TM/saline,TM/early EPC,0.2,0.4,0.8,1.0,0.6,Perfusion Ratio,0,1.2,7,21,0,(days),Analysis of Perfusion in Ischemic Limbs,All p0.05,*,*,*,*,TM + early EPC,M-PECAM-1 / Laminin,PBS/Saline,TMD23/Saline,TMD23/eEPC,PBS/eEPC,Capillary density in calf of hin
17、dlimb ischemic mice treated with early EPC + TMD23,Capillary / myocyte density,50 mm,hNA,Thigh tissue of hindlimb ischemic mice treated with early EPC + TMD23 (21st days),H33258/hNA,H33258,hNA/h-CD31,hNA/vWF,20,40,60,80,100,Normal calf,Saline,Saline,Early EPC,Early EPC,PBS,TMD23,Muscle weigh ( mg ),
18、*,*,*,Pump:,#,#,#,#,# p0.05 compared with normal calf * p0.05 compared with PBS,Calf muscle in hindlimb ischemic mice treated with TMD23 and early EPC,Summary,Human early EPC express and release TM, the amount of which increased along the initial 21 days of culture TMD23 increases CFU of cultured PB
19、MNC TMD23 and TMD123 fragments possess chemotaxic effects on early EPC TMD23 increases angiopoietin 2 expression in early EPC. TMD23 increases cytoplasmic MMP-9 activity in early EPC TMD23 activates ERK 1/2 and Akt In mouse ischemic hindlimb treated with early EPC, TMD23 enhances subcutaneous perfus
20、ion, increases capillary density, and lessens limb loss,Conclusion,TM is generated and released by early EPC Exogenous TMD23 enhances the angiogenic potential of early EPC Co-dministration of TMD23 plus early EPC has therapeutic potential in peripheral artery disease.,Thanks for your attention,馬偕紀念醫院淡水院區 馬偕樓,
