辛伐他汀对内皮祖细胞增殖、迁移、凋亡及黏附影响的研究
摘要
目的:细胞水平观察不同浓度辛伐他汀对内皮祖细胞(endothelialprogenitor cells,EPCs)功能的影响。
方法:采用密度梯度离心法从人外周血获取单个核细胞,将其接种在人纤维连接蛋白包被培养板上,培养7天后,收集贴壁细胞,通过激光共聚焦显微镜鉴定,FITC-UEA-I和DiI-acLDL双染色阳性细胞为EPCs。以不同浓度辛伐他汀(分别为0.0001、0.001、0.01、0.1、1.0μM)或0.01μM辛伐他汀+PI3K阻断剂(LY294002)和EPCs培养24h。分别采用MTT比色法、改良的Boyden小室、流式细胞仪和黏附能力测定实验来观察辛伐他汀对EPCs的增殖、迁移、凋亡和黏附影响,并探讨LY294002干预作用。
结果:辛伐他汀显著提高了外周血EPCs的增殖能力、迁移能力、抗凋亡能力和黏附能力(P＜0.05)。辛伐他汀浓度在0.01μM时对EPCs功能影响达到最大。随着药物浓度的继续增大,EPCs的上述功能反呈下降趋势,但0.1μM组仍高于对照组。辛伐他汀对EPCs的功能影响能被LY294002(PI3K抑制剂)阻断。
结论:辛伐他汀能增加EPCs的增殖、迁移、抗凋亡和黏附能力,其机制可能与PI3K/Akt信号途径有关。
Objective: To investigate in vitro whether simvastatins of different concentrations have influences on the functions of endothelial progenitor cells (EPCs).
Methods: Total mononuclear cells (MNCs) were isolated from human peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days cultured, attached cells were isolated and were assessed with a laser scanning confocal microscope, EPCs were characterized as cells double positive for DiLDL-uptake and lectin binding. EPCs were treated with simvastatins of different concentrations (0.0001、0.001、0.01、0.1、1.0μM),or LY294002(PI3K inhibitor)+ simvastatins(0.01μM) for 24h. The influences of Simvastatins on EPCs' proliferation, migration , apoptosis were assayed with MTT assay, modified Boyden chamber assay and FACS assay, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then the adherent cells were counted.
Results: Simvastatins strikingly improved the ability in proliferating, migrating, anti-apoptosis and adhering of EPCs isolated from human peripheral blood (p<0.05). Simvastatins at 0.01μM concentration reached the maximum effect on EPCs. While the concentration of simvastatins was further increased more than 0.01μM, the above improvement effects showed a tendency to decline, but the effects at 0.1μM concentration were still greater than those in control group. And the influence of Simvastatins on these functions of EPCs could be abolished by LY294002(PI3K inhibitor).
Conclusion: The present study established that Simvastatins could improve EPCs' proliferation, migration , anti-apoptosis and adhesion and the mechanism maybe involves the PI3K/AKt pathway.
方法:采用密度梯度离心法从人外周血获取单个核细胞,将其接种在人纤维连接蛋白包被培养板上,培养7天后,收集贴壁细胞,通过激光共聚焦显微镜鉴定,FITC-UEA-I和DiI-acLDL双染色阳性细胞为EPCs。以不同浓度辛伐他汀(分别为0.0001、0.001、0.01、0.1、1.0μM)或0.01μM辛伐他汀+PI3K阻断剂(LY294002)和EPCs培养24h。分别采用MTT比色法、改良的Boyden小室、流式细胞仪和黏附能力测定实验来观察辛伐他汀对EPCs的增殖、迁移、凋亡和黏附影响,并探讨LY294002干预作用。
结果:辛伐他汀显著提高了外周血EPCs的增殖能力、迁移能力、抗凋亡能力和黏附能力(P＜0.05)。辛伐他汀浓度在0.01μM时对EPCs功能影响达到最大。随着药物浓度的继续增大,EPCs的上述功能反呈下降趋势,但0.1μM组仍高于对照组。辛伐他汀对EPCs的功能影响能被LY294002(PI3K抑制剂)阻断。
结论:辛伐他汀能增加EPCs的增殖、迁移、抗凋亡和黏附能力,其机制可能与PI3K/Akt信号途径有关。
Objective: To investigate in vitro whether simvastatins of different concentrations have influences on the functions of endothelial progenitor cells (EPCs).
Methods: Total mononuclear cells (MNCs) were isolated from human peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days cultured, attached cells were isolated and were assessed with a laser scanning confocal microscope, EPCs were characterized as cells double positive for DiLDL-uptake and lectin binding. EPCs were treated with simvastatins of different concentrations (0.0001、0.001、0.01、0.1、1.0μM),or LY294002(PI3K inhibitor)+ simvastatins(0.01μM) for 24h. The influences of Simvastatins on EPCs' proliferation, migration , apoptosis were assayed with MTT assay, modified Boyden chamber assay and FACS assay, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then the adherent cells were counted.
Results: Simvastatins strikingly improved the ability in proliferating, migrating, anti-apoptosis and adhering of EPCs isolated from human peripheral blood (p<0.05). Simvastatins at 0.01μM concentration reached the maximum effect on EPCs. While the concentration of simvastatins was further increased more than 0.01μM, the above improvement effects showed a tendency to decline, but the effects at 0.1μM concentration were still greater than those in control group. And the influence of Simvastatins on these functions of EPCs could be abolished by LY294002(PI3K inhibitor).
Conclusion: The present study established that Simvastatins could improve EPCs' proliferation, migration , anti-apoptosis and adhesion and the mechanism maybe involves the PI3K/AKt pathway.
引文
[1]Rafii S,Lyden D.Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration[J].Nat Med,2003,9:702-712.
[2]Risau,W.Mechanisms of angiogenesis[J].Nature,1997,386:671-674.
[3]Carmeliet,P,and Jain,R.K.Angiogenesis in cancer and other diseases[J].Nature,2000,407:249-257.
[4]丁志坚,张宇燕,张馥敏,等.心肌梗死患者自体骨髓干细胞移植后近、中期心肌代谢及收缩功能的变化[J].南京医科大学学报2006,26(11):1086-1089,1102.
[5]Kawamoto A,Tkebuchava T,Yamaguchi J,et al.Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia[J].Circulation,2003,107:461-468.
[6]Maron DJ,Fazio S,Linton MF.Current perspectives on statins[J].Circulation,2000,101:207-213.
[7]Sacks F.M,Pfeffer MA,Moye LA,et al.The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels.Cholesterol and Recurrent Events Trial investigators[J].N Engl J Med,1996,335:1001-1009.
[8]Hill JM,Zalos G,Halcox JP,et al.Circulating endothelial progenitor cells,vascular function,and cardiovascular risk[J].N Engl J Med,2003,348:593-600.
[9] Vasa M, Fichtlseherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J].Circ Res,2001,89:El-7.
[10] C J Newton, G Ran, Y-X Xie, et al.Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone[J]. Journal of Endocrinology, 2002, 174: 7-16.
[11] Walter DH, Rittig K, Bahlmann FH, et al.Statin therapy accelerate reendothelialization[J].Circulation,2002,105:3017-3024.
[12] Murohara T, Tepper O, Silver M, et al. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization[J]. Exp Hematol, 2002,30:967.
[13] Suzuki T. Nishida M. Futami S, et al.Neoendothelialization after peripheral blood stem cell transplantation in humans[J]. A case report of a Tokaimura nuclear accident victim[J]. Cardiovasc Res,2003,58:487-492.
[14] Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury[J].CircRes,2003,93(2):e17-e24.
[15] Griese DP, Melo LG, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy [J]. Circulation,2003,108(21):2710-2715.
[16]Llevadot J, Murasawa S, Kureishi Y, et al. HMG-CoA reductase inhibitor mobilizes bone marrow-derived endothelial progenitor cells [J]. J Clin Invest,2001, 108:399-405.
[17] Chang, H.W., et al. Transformation of chicken cells by the gene encoding the catalytic subunit of PI3-kinase[J]. Science, 1997,276:1848- 1850.
[18] Khwaja, A., Rodriguez-Viciana, P., Wennstrom, S., Warne, P.H., and Downward, J. Matrix adhesion and ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase b/akt cellular survival pathway [J]. EMBO J, 1997, 16:2783-2793.
[19] Franke, T.F., Kaplan, D.R., Cantley, L.C., and Toker, A. Direct regulation of the akt proto-oncogene product by phosphatidylinositol-3,4-bishosphate[J]. Science, 1997,275:665-668.
[20] Eves, E.M., et al. Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line[J]. Mol Cell Biol, 1998,18:2143-2152.
[21] Crowder, R.J., and Freeman, R.S. Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons[J]. J Neurosci, 1998,18:2933-2943.
[22] Ulrich, E., et al. Specific TrkA survival signals interfere with different apoptotic pathways[J]. Oncogene, 1998, 16:825-832.
[23] Dimmeler, S., and Zeiher, A.M. Akt takes center stage in angiogenesis signaling[J].2000, Circ Res, 86:4-5.
[24] Gerber, H.P., et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'- kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation[J]. J Biol Chem, 1998,273:30336-30343.
[25] Kontos, C.D., et al. Tyrosine 1101 of tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3- kinase and akt[J]. Mol Cell Biol, 1998,18:4131-4140.
[26] Kim, I., et al. Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/akt signal transduction pathway[J]. Circ Res, 2000,86:24-29.
[27] Papapetropoulos, A., et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the akt/survivin pathway [J]. J Biol Chem, 2000,275:9102-9105.
[28] Carmeliet, P., et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis [J]. Cell, 1999, 98:147-157.
[29] Fujio, Y., Nguyen, T., Wencker, D., Kitsis, R.N., and Walshi, K. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart[J]. Circulation, 2000,101:660- 667.
[30] Ioakim Spyridopoulos, Judith Haendeler, et al.Statins Enhance Migratory Capacity by Upregulation of the Telomere Repeat-Binding Factor TRF2 in Endothelial Progenitor Cells[J]. Circulation, 2004,110:3136-3142.
[1]丁志坚,张宇燕,张馥敏,等 心肌梗死患者自体骨髓干细胞移植后近、中期心肌代谢及收缩功能的变化[J].南京医科大学学报 2006,26(11):1086-1089,1102.
[2]Kawamoto A,Tkebuchava T,Yamaguchi J,et al..Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia[J].Circulation,2003,107:461- 468.
[3]Suda T,Takakura N,Oike Y.Hematopoiesis and angiogenesis[J].Int J Hematol,2000,71(2):99-107.
[4]Asahara T,Murohara T,Sullivan A,et al.Isolation of putative progenitor enddothelial cells for angiogenesis[J].Science,1997,275:964-967.
[5]Shmelkov,L Jun,et al.Alternative promoters regulate transcription of the gene that encodes stem cell surface protein AC133[J].Blood,2004,103(6):2055-2061.
[6]Walter DH,Dimmeler S.Endothelial progenitor cells:regulation and contribution to adult neovascularization.Herz.2002;27:579-588.
[7]Juliane E,Gergely J et al.Endothelial progenitor cell culture and differentiation in vitro:a methodological comparison using human umbilical cord blood[J].Cardiovasc Res,2003,58(02):478-486.
[8]Hur J,Yoon CH,Kim HS,et al.Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis[J]. Arterioscler Thromb Vasciol,2004,24(2): 288-293.
[9] Badorff C, Brandes RP, Popp R, et al. Transdiferentiation of blood derived human adult endothelial progenitor cells into functionally active cardiomyocytes[J]. Circulation,2003,107(7): 1024-1032.
[10] Mario Peichev, Afzal J. Naiyer, et al.Expression of VEGFR-2 and AC133 by circulating human CD34~+ cells identifies a population of functional endothelial precursors[J]. Blood, 2000,95: 952 - 958.
[11] Vasa M, Fichtlseherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J].Circ Res,2001,89: E1-7
[12] Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[J]. Nat Med, 1999 ,5:434 -438.
[13] Hill JM, Zalos G, Haleox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk[J]. N Engl J Med,2003,348(7):593-600.
[14] Choi JH, Kim KL, Huh W, et al .Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure [J] .Arterioscler Thromb Vasc Biol,2004,24(7): 1246-1252.
[15] Imanishi T, Hano T, Sawanmra T, et al. Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction[J].Clin Exp Pharmacol Physiol,2004,31(7): 407-413.
[16] Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin Of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization[J] .Circ Res, 1999,85: 221-228.
[17] Jun-ichi Yamaguchi, Kengo Fukushima Kusano, Osamu Masuo et al.Stromal Cell-Derived Factor-1 Effects on Ex Vivo Expanded Endothelial Progenitor Cell Recruitment for Ischemic Neovascularization [J]. Circulation,2003,107:1322-1328.
[18] Gill M, Dia S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2+AC133+ endothelial precursor cells [J].Circ Res,2001,88:167-174.
[19] DH. Walter, Kilian Rittig, Ferdinand H,et al. Statin Therapy Accelerates Reendothelialization: A Novel Effect Involving Mobilization and Incorporation of Bone Marrow-Derived Endothelial Progenitor Cells [J].Circulation,2002,105:3017-3024.
[20] Sata M,Saiura A,Kunisato A,et al.Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis Of atherosclerosis[J].NatureMedicine,2002,8:403-407.
[21]IsnerJ M , Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization[J]. J Clin Invest, 1999, 103(9):1231-1236.
[22]Gulati R, Jevremovie D,Peterson TE,et al.Autologous culture-modified mononuclear cells confer vascular protection after arterial injury[J].Circulation,2003,108(12): 1520-1526.
[23] Werner N,Junk S,Laufs U,et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury[J]. Circ Res,2003,93(2):e17-e24.
[24]Tateishi-Yuyama E,Matsubara H,Murohara T,et al.Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: pilot study and a randomized controlled trial[J].Lancet,2002,360:427-435.
[25]Urbich C ,Heeschen C ,Aicher A,et al.Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells[J].Circulation,2003,108:2511-2516.
[26]Kang HJ,Kim HS,Zhang SY,et al .Effects of intracoronary infusion of peripheral blood stem cells mobilized with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction:the MAGIC cell randomized clinical trial[J].Lancet,2004,363(9411):751-756.
[27]Kamihata, H, Matsuhara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines [J]. circulation, 2001,104:1046-1052.
[28]Strauer BE, Brehm M, Zeus T, et al Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans[J].Circulation, 2002,106:1913-1918.
[29]Assmus B, Schachinger V, Teupe C, et al.Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI)[J]. circulation, 2002,106:3009-3017.
[30]Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone marrow cell transfer after myocardial infarction:the BOOST randomized controlled clinical trial[J].Lancet,2004,364:141-148.
[31]Kong D,Melo LG, Mangi AA, et al. Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells[J].Circulation, 2004,109:1769-1775.
[2]Risau,W.Mechanisms of angiogenesis[J].Nature,1997,386:671-674.
[3]Carmeliet,P,and Jain,R.K.Angiogenesis in cancer and other diseases[J].Nature,2000,407:249-257.
[4]丁志坚,张宇燕,张馥敏,等.心肌梗死患者自体骨髓干细胞移植后近、中期心肌代谢及收缩功能的变化[J].南京医科大学学报2006,26(11):1086-1089,1102.
[5]Kawamoto A,Tkebuchava T,Yamaguchi J,et al.Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia[J].Circulation,2003,107:461-468.
[6]Maron DJ,Fazio S,Linton MF.Current perspectives on statins[J].Circulation,2000,101:207-213.
[7]Sacks F.M,Pfeffer MA,Moye LA,et al.The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels.Cholesterol and Recurrent Events Trial investigators[J].N Engl J Med,1996,335:1001-1009.
[8]Hill JM,Zalos G,Halcox JP,et al.Circulating endothelial progenitor cells,vascular function,and cardiovascular risk[J].N Engl J Med,2003,348:593-600.
[9] Vasa M, Fichtlseherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J].Circ Res,2001,89:El-7.
[10] C J Newton, G Ran, Y-X Xie, et al.Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone[J]. Journal of Endocrinology, 2002, 174: 7-16.
[11] Walter DH, Rittig K, Bahlmann FH, et al.Statin therapy accelerate reendothelialization[J].Circulation,2002,105:3017-3024.
[12] Murohara T, Tepper O, Silver M, et al. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization[J]. Exp Hematol, 2002,30:967.
[13] Suzuki T. Nishida M. Futami S, et al.Neoendothelialization after peripheral blood stem cell transplantation in humans[J]. A case report of a Tokaimura nuclear accident victim[J]. Cardiovasc Res,2003,58:487-492.
[14] Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury[J].CircRes,2003,93(2):e17-e24.
[15] Griese DP, Melo LG, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy [J]. Circulation,2003,108(21):2710-2715.
[16]Llevadot J, Murasawa S, Kureishi Y, et al. HMG-CoA reductase inhibitor mobilizes bone marrow-derived endothelial progenitor cells [J]. J Clin Invest,2001, 108:399-405.
[17] Chang, H.W., et al. Transformation of chicken cells by the gene encoding the catalytic subunit of PI3-kinase[J]. Science, 1997,276:1848- 1850.
[18] Khwaja, A., Rodriguez-Viciana, P., Wennstrom, S., Warne, P.H., and Downward, J. Matrix adhesion and ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase b/akt cellular survival pathway [J]. EMBO J, 1997, 16:2783-2793.
[19] Franke, T.F., Kaplan, D.R., Cantley, L.C., and Toker, A. Direct regulation of the akt proto-oncogene product by phosphatidylinositol-3,4-bishosphate[J]. Science, 1997,275:665-668.
[20] Eves, E.M., et al. Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line[J]. Mol Cell Biol, 1998,18:2143-2152.
[21] Crowder, R.J., and Freeman, R.S. Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons[J]. J Neurosci, 1998,18:2933-2943.
[22] Ulrich, E., et al. Specific TrkA survival signals interfere with different apoptotic pathways[J]. Oncogene, 1998, 16:825-832.
[23] Dimmeler, S., and Zeiher, A.M. Akt takes center stage in angiogenesis signaling[J].2000, Circ Res, 86:4-5.
[24] Gerber, H.P., et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'- kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation[J]. J Biol Chem, 1998,273:30336-30343.
[25] Kontos, C.D., et al. Tyrosine 1101 of tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3- kinase and akt[J]. Mol Cell Biol, 1998,18:4131-4140.
[26] Kim, I., et al. Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/akt signal transduction pathway[J]. Circ Res, 2000,86:24-29.
[27] Papapetropoulos, A., et al. Angiopoietin-1 inhibits endothelial cell apoptosis via the akt/survivin pathway [J]. J Biol Chem, 2000,275:9102-9105.
[28] Carmeliet, P., et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis [J]. Cell, 1999, 98:147-157.
[29] Fujio, Y., Nguyen, T., Wencker, D., Kitsis, R.N., and Walshi, K. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart[J]. Circulation, 2000,101:660- 667.
[30] Ioakim Spyridopoulos, Judith Haendeler, et al.Statins Enhance Migratory Capacity by Upregulation of the Telomere Repeat-Binding Factor TRF2 in Endothelial Progenitor Cells[J]. Circulation, 2004,110:3136-3142.
[1]丁志坚,张宇燕,张馥敏,等 心肌梗死患者自体骨髓干细胞移植后近、中期心肌代谢及收缩功能的变化[J].南京医科大学学报 2006,26(11):1086-1089,1102.
[2]Kawamoto A,Tkebuchava T,Yamaguchi J,et al..Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia[J].Circulation,2003,107:461- 468.
[3]Suda T,Takakura N,Oike Y.Hematopoiesis and angiogenesis[J].Int J Hematol,2000,71(2):99-107.
[4]Asahara T,Murohara T,Sullivan A,et al.Isolation of putative progenitor enddothelial cells for angiogenesis[J].Science,1997,275:964-967.
[5]Shmelkov,L Jun,et al.Alternative promoters regulate transcription of the gene that encodes stem cell surface protein AC133[J].Blood,2004,103(6):2055-2061.
[6]Walter DH,Dimmeler S.Endothelial progenitor cells:regulation and contribution to adult neovascularization.Herz.2002;27:579-588.
[7]Juliane E,Gergely J et al.Endothelial progenitor cell culture and differentiation in vitro:a methodological comparison using human umbilical cord blood[J].Cardiovasc Res,2003,58(02):478-486.
[8]Hur J,Yoon CH,Kim HS,et al.Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis[J]. Arterioscler Thromb Vasciol,2004,24(2): 288-293.
[9] Badorff C, Brandes RP, Popp R, et al. Transdiferentiation of blood derived human adult endothelial progenitor cells into functionally active cardiomyocytes[J]. Circulation,2003,107(7): 1024-1032.
[10] Mario Peichev, Afzal J. Naiyer, et al.Expression of VEGFR-2 and AC133 by circulating human CD34~+ cells identifies a population of functional endothelial precursors[J]. Blood, 2000,95: 952 - 958.
[11] Vasa M, Fichtlseherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[J].Circ Res,2001,89: E1-7
[12] Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization[J]. Nat Med, 1999 ,5:434 -438.
[13] Hill JM, Zalos G, Haleox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk[J]. N Engl J Med,2003,348(7):593-600.
[14] Choi JH, Kim KL, Huh W, et al .Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure [J] .Arterioscler Thromb Vasc Biol,2004,24(7): 1246-1252.
[15] Imanishi T, Hano T, Sawanmra T, et al. Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction[J].Clin Exp Pharmacol Physiol,2004,31(7): 407-413.
[16] Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin Of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization[J] .Circ Res, 1999,85: 221-228.
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