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）

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 lower 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 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 2828 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, TcO2 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 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 mononuclear 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 cobblestoneShape 7 days 2-3 weeksPeak growth less less less more more more more less Surface marker VE-cadherin Flt-1 KDR CD45 Angiogenic cytokines VEGF IL-8 more more less less less moreNO production unable ableCapillary tube formation Modified from Yoon et al. Circulation 2005 and Hur J et al. ATVB 2004 heterogeneous homogeneousPopulations good goodAngiogenic 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 fragments 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 human PBMNC cultured in different medium EndoCult EGM2 PBMC HAEC Day 7 Day 7 Day 21Day 21 50 mm 80 20 40 60 0 100 TM Conc. (pg/ mL ) Day 7 Day 14 Day 21Medium only All p0.05 TM is released by cultured human PBMNC CFU in PBMNC respectively treated with TMD1, TMD23 and TMD123 (100 ng/ml) CFU number/ 1x10 6 PBMC P 0.05 Ctrl TMD1 TMD23 TMD123 Chemotaxic effects of TMD (100 ng/ml) on human early EPC 300 mm Ctrl TMD123TMD23 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 treated with TMD23 (100 ng/ml) for 72 hour Ctrl TMD 23 Ctrl TMD 23 Medium HAEC PMA 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 MMP9 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 Grou p Osmoti c pump Artery ligation Injected cells 1 PBS + Saline 2 PBS + Early EPC 3 TMD23 + Saline 4 TMD23 + Early EPC 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 flow 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 EPCEarly EPC TMD23PBS 8 88 8 Pump: % n= Amputation Toe necrosis Limb salvage 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 7 210 (days) 0 days 7 days 21 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 hindlimb ischemic mice treated with early EPC + TMD23 p0.05 compared with PBS # p0.05 compared with TM/ eEPC * Capillary / myocyte density Saline PBS TMD23Pump: 0.5 1.0 1.5 2.0 * * *# # * Saline Early EPC Early EPC 50 mm hNA Thigh tissue of hindlimb ischemic mice treated with early EPC + TMD23 (21st days) H33258/hNAH33258 hNA/h-CD31 hNA/vWF 20 40 60 80 100 Normal calfSaline SalineEarly EPCEarly EPC PBS TMD23 Muscle weigh ( mg ) * * * 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