摘要
目的
研究人胚胎血管内皮祖细胞（endothelial progenitor cells，EPCs）分离、培养及鉴定的方法，评价其用于治疗动脉粥样硬化的可行性,为干细胞技术治疗疾病提供可靠的细胞来源。
方法
1.从8-14周龄流产的人胚胎主动脉中应用胶原酶消化法分离获得EPCs，用含有碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)、表皮生长因子(epidermal growth factor,EGF)和白血病抑制因子(leucocyte inhibitory factor,LIF)的低血清培养基体外扩增培养EPCs。分离培养的EPCs鉴定采用细胞免疫荧光染色、RT-PCR及流式细胞术，检测EPCs细胞的特异标志CD133、CD34和血管内皮细胞生长因子受体2（vascular endothelial growth factor receptor2,VEGFR2）。培养的EPCs应用VEGF进行诱导分化，并评价其分化成为血管内皮细胞的能力。
2.将用绿色荧光蛋白（green fluorescent protein，GFP）标记的EPCs通过尾静脉移植到apoE基因敲除小鼠动脉粥样硬化模型中，24h后取出小鼠主动脉，镜下观察动脉粥样硬化病变内膜EPCs的定位粘附情况。
结果
1.分离的人胚胎主动脉EPCs表达EPCs的标志分子CD133，CD34和VEGFR2。EPCs在体外特定低血清培养条件下表现很强的增殖能力，并具有摄取Ac-LDL和体外成管能力。培养的EPCs经过VEGF诱导后，细胞表达CD133明显降低，表达vWF，CD31和ELAM-1增强，表明细胞分化成为血管内皮细胞。
2.成功构建动脉粥样硬化模型，并在此动脉粥样硬化模型中，检测到有人胚胎主动脉EPCs定位并粘附到粥样硬化病变内膜。
结论
人胚胎早期主动脉的EPCs具有很好的体外自我更新能力和分化成为血管内皮细胞的潜能；EPCs可以粘附到apoE基因敲除小鼠主动脉粥样硬化病变部位，具有内皮修复的潜能，可以用作治疗动脉粥样硬化疾病的细胞材料。
Objective
To evaluate the feasibility of human fetal aorta-derived EPCs transfusion for thetreatment of atherosclerotic lesions, we isolated, cultured and identified endothelialprogenitor cells (EPCs) from human fetal aorta. The capability of EPCs differentiating intoendothelial cells was also analyzed. And then we evaluated the role of intravenouslytransfused human fetal aorta-derived EPCs on atherosclerotic lesions in apolipoprotein Eknockout(apoE-/-) mice.
Methods
1. EPCs were isolated from clinical aborted fetal aorta by digesting aorta with type Icollagenase. EPCs were cultured in DMEM/F12medium supplied with LIF, bFGF, EGF,ECGS and low serum. The expression of CD133, CD34and VEGFR2was assessed byRT-PCR, immunofluorescence staining and FACS analysis. EPCs were induced towardendothelial cells by cultured with medium containing20%FCS and VEGF.
2. With ApoE-/-mice, which is prone to developing atherosclerotic lesions acceleratedby high-fat diet feeding,we attempted to investigate whether the GFP-labeled EPCs canhome to the injured endothelium and whether the local atherosclerosis formation can beprevented by such EPCs administration.
Results
1. EPCs derived from human fetal aorta expressed CD133, CD34and VEGFR2. TheEPCs proliferated rapidly in DMEM/F12containing LIF, bFGF, EGF and ECGS. EPCspossessed the ability of uptaking DiI-Ac-LDL and tube formation on matrigel. After induced with VEGF, the expression of CD133was decreased significantly; and theexpression of vWF, CD31and ELAM-1was increased.
2. We established mice atherosclerosis model Successfully. We found investigate theGFP-labeled EPCs homing to the injured endothelium and the local atherosclerosisformation can be prevented by such EPCs administration..
Conclusions
EPCs derived from early fetal aorta possess strong capability of self-renew and thepotential of differentiating into endothelial cells. EPCs may play an important role inrestoration of endothelial injury and prevention of atherosclerosis, which indicate thesecells may have the potential value for the treatment of atherosclerotic lesions.
引文
[1] Mangge H，Almer G, Truschnig-Wilders M, et al. Inflammation, adiponection,obesityand cardiovascular risk. Curr Med Chem.2010;17(36):4511-4520
[2]阮秋蓉.干细胞/祖细胞与动脉粥样硬化研究进展.中国动脉硬化杂志.2011;19(5):375-379.
[3] Libby, P. Inflammation in atherosclerosis. Nature.2002;420:868–874.
[4] Ross, R. Atherosclerosis: an inflammatory disease. N. Engl. J. Med.1999;340:115–126.
[5] Lusis AJ. Atherosclerosis. Nature.2000;407(6801):233-241.
[6] Rauscher FM, Goldchmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cellexhaustion, and atherosclerosis. Circulation.2003;108(4):457-463.
[7] Shirota, T, H. Yasui, Shimokawa, H. et al. Fabrication of endothelial progenitor cell(EPC)-seeded intravascular stent devices and in vitro endothelialization on hybridvascular tissue. Biomsterials.2003;24(13):2295-2302.
[8] Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitorcells reduces neointima formation after vascular injury. Circ Res.2003;93: e17–e24.
[9] Foteinos G, Hu Y, Xiao Q, et al. Rapid endothelial turnover in atherosclerosis-proneareas coincides with stem cell repair in apolipoprotein E deficient mice. Circulation.2008;117:1856–1863.
[10] Fujiyama S, Amano K, Uehira K, et al. Bone marrow monocyte lineage cells adhereon injured endothelium in a monocyte chemoattractant protein-1-dependent mannerand accelerate reendothelialization as endothelial progenitor cells. Circ Res.2003;93:980–989.
[11] Griese DP, Ehsan A, Melo LG, et al. Isolation and transplantation of autologouscirculating endothelial cells into denuded vessels and prosthetic grafts: Implicationsfor cell-based vascular therapy. Circulation.2003;108:2710–2715.
[12] Gulati R, Jevremovic D, Peterson TE, et al. Autologous culture-modified mononuclearcells confer vascular protection after arterial injury.Circulation.2003;108:1520–1526.
[13] He T, Smith LA, Harrington S, et al. Transplantation of circulating endothelialprogenitor cells restores endothelial function of denuded rabbit carotid arteries.Stroke.2004;35:2378–2384.
[14] Fadini, GP, Coracina, A, Baesso, I. et al. Peripheral blood CD34+KDR+endothelialprogenitor cells are determinants of subclinical atherosclerosis in a middle-agedgeneral population. Stroke.2006;37(9): p2277-2282.
[15] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelialcells for angiogenesis. Science.1997;275(5302):964-7.
[16] Suda T, Takakura N, Oike Y. Hematopoiesis and angiogenesis. International journalof hematology.2000;71(2):99-107.
[17] Alessandri, G., M. Girelli, et al. Human vasculogenesis ex vivo: embryonal aorta as atool for isolation of endothelial cell progenitors. Lab Invest.2001;81(6):875-885.
[18] Invernici G,Emanueli C,Madeddu P,et al. Human fetal aorta contains vascularprogenitor cells capable of inducing vasculogenesis, angiogenesis, and myogenesis invitro and in a murine model of peripheral ischemia.Am J Pathol.2007;170(6):1879-1892.
[19] Ma ZL，Mai XL，Sun JH, et al. Inhibited atherosclerotic plaque formation by localadministration of magnetically labeled endothelial progenitor cells (EPCs) in a rabbitmodel. Atherosclerosis.2009;205(1):80–86.
[20] Kawamoto A, Tkebuchava T, Yamaguchi J, et al. Intramyocardial transplantation ofautologous endothelial progenitor cells for therapeutic neovascularization ofmyocardial ischemia. Circulation.2003;107(3):461-468.
[21] Briasoulis A, Tousoulis D, Antoniades C, et al.The Role of Endothelial ProgenitorCells in Vascular Repair after Arterial Injury and Atherosclerotic PlaqueDevelopment.Cardiovascular Therapeutics.2010:1–15.
[22] Urbich C, Heeschen C, Aicher A, et al.Relevance of monocytic features forneovascularization capacity of circulating endothelial progenitor cells. Circulation.2003;108(20):2511-2516.
[23] Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelialprogenitor cells responsible for postnatal vasculogenesis in physiological andpathological neovascularization.Circ Res.1999;85(3):221-228.
[24] Benameur T, Tual-Chalot S, Andriantsitohaina R, et al. PPARalpha is essential formicroparticle-induced differentiation of mouse bone marrow-derived endothelialprogenitor cells and angiogenesis. PLoS One.2010;5(8): e12392.
[25] Ahrens I, Domeij H, Topcic D, et al. Successful in vitro expansion and differentiationof cord blood derived CD34+cells into early endothelial progenitor cells revealshighly differential gene expression. PLoS One.2011;6(8): e23210.
[26] Rehman J, Li J, Orschell CM, et al. Peripheral blood "endothelial progenitor cells" arederived from monocyte/macrophages and secrete angiogenic growth factors.Circulation.2003;107(8):1164-1169.
[27] Caiado F, Carvalho T, Silva F, et al. The role of fibrin E on the modulation ofendothelial progenitors adhesion, differentiation and angiogenic growth factorproduction and the promotion of wound healing. Biomaterials.2011;32(29):7096-105.
[28] Lee MJ, Kim J, Lee KI, et al. Enhancement of wound healing by secretory factors ofendothelial precursor cells derived from human embryonic stem cells. Cytotherapy.2011;13(2):165-178.
[29] Alev C, Ii M, Asahara T. Endothelial Progenitor Cells: a novel tool for the therapy ofischemic diseases. Antioxidants&Redox Signaling.2011;15(4):949-965.
[30] Kocaman S A, Yalcin M R, Yagci M, et a1.Endothelial progenitor cells(CD34+KDR+) and monocytes may provide the development of good coronarycollaterals despite the vascular risk factors and extensive atherosclerosis. AnadoluKardiyol Derg.2011;11(4):290-299.
[31] Yoder MC. Defining human endothelial progenitor cells. J Thromb Haemost.2009;7Suppl1:49-52.
[32] Eggermann J, Kliche S, Jarmy G, et a1.Endothelial progenitor cell culture anddifferentiation in vitro:a methodological comparison using human umbilical cordblood.Cardiovasc Res.2003;58(2):478-486.
[33] Yin AH, Miraglia S, Zanjani ED, et al. AC133,a novel marker for humanhematopoietic stem and progenitor cells. Blood.1997;90(12):5002-5012.
[34] Grau GE, Mili N, Lou JN, et al. Phenotypic and functional analysis of pulmonarymicrovascular endothelial cells from patients with acute respiratory distress syndrome.Lab Invest.1996;74(4):761-770.
[1] Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cellexhaustion, and atherosclerosis. Circulation.2003;29;108(4):457-463.
[2]阮秋蓉.干细胞/祖细胞与动脉粥样硬化研究进展.中国动脉硬化杂志.2011;19(5):375-379.
[3] Bahlmann FH, de Groot K, Mueller O, et al. Stimulationof endothelial progenitorcells: A new putative therapeutic effect of angiotensin II receptor antagonists.Hypertension.2005;45:526–529.
[4] Chen JZ, Zhang FR, Tao QM, et al. Number and activity of endothelial progenitor cellsfrom peripheral blood in patients with hypercholesterolaemia. Clin Sci (Lond).2004;107:273–280.
[5] Chen YH, Lin SJ, Lin FY, et al. High glucose impairs early and late endothelialprogenitor cells by modifying nitric oxide-related but not oxidative stress-mediatedmechanisms. Diabetes.2007;56:1559–1568.
[6] Laufs U, Wassmann S, Czech T, et al. Physical inactivity increases oxidative stress,endothelial dysfunction, and atherosclerosis. Arterioscler Thromb Vasc Biol.2005;25:809–814.
[7] Laufs U, Werner N, Link A, et al. Physical training increases endothelial progenitorcells, inhibits neointima formation, and enhances angiogenesis. Circulation.2004;109:220–226.
[8] Pellegatta F, Bragheri M, Grigore L, et al. In vitro isolation of circulating endothelialprogenitor cells is related to the high density lipoprotein plasma levels. Int JMolMed.2006;17:203–208.
[9] Dimmeler S, Aicher A, Vasa M, et al. HMG-CoA reductase inhibitors (statins) increaseendothelial progenitor cells via the PI3-kinase/Akt pathway. J Clin Invest.2001;108:391–397.
[10] Iwakura A, Luedemann C, Shastry S, et al. Estrogen-mediated, endothelial nitric oxidesynthase-dependent mobilization of bone marrow-derived endothelial progenitor cellscontributes to reendothelialization after arterial injury. Circulation.2003;108:3115–3121.
[11] Werner N, Junk S, Laufs U, et al. Intravenous transfusion of endothelial progenitorcells reduces neointima formation after vascular injury. Circ Res.2003;93: e17–e24.
[12] Foteinos G, Hu Y, Xiao Q, et al. Rapid endothelial turnover in Atherosclerosis proneareas coincides with stem cell repair in apolipoprotein E deficient mice.Circulation.2008;117:1856–1863.
[13] Fujiyama S, Amano K, Uehira K, et al. Bone marrow monocyte lineage cells adhereon injured endothelium in a monocyte chemoattractant protein-1-dependent mannerand accelerate reendothelialization as endothelial progenitor cells. Circ Res.2003;93:980–989.
[14] Griese DP, Ehsan A, Melo LG, et al. Isolation and transplantation of autologouscirculating endothelial cells into denuded vessels and prosthetic grafts: Implicationsfor cell-based vascular therapy.Circulation.2003;108:2710–2715.
[15] Gulati R, Jevremovic D, Peterson TE, et al. Autologous culture-modied mononuclearcells confer vascular protection after arterial injury. Circulation.2003;108:1520–1526.
[16] He T, Smith LA, Harrington S, et al. Transplantation of circulating endothelialprogenitor cells restores endothelial function of denuded rabbit carotid arteries.Stroke.2004;35:2378–2384.
[17]司逸,符伟国,竺挺等.内皮祖细胞促动脉内皮损伤修复的研究.中国分子心脏病学杂志.2010;10（1）：17-22
[18]刘楠梅,田军,程劲胡等.骨髓间充质干细胞向急性肾损伤小鼠的肾脏归巢现象及功能探讨.肾脏病与透析移植杂志.2009;18（5）：430-437
[19] Ara T, Tokoyoda K, Okamoto R, et al. The role of CXCL12in the organ-specificprocess of artery formation. Blood.2005;105:3155–3161.
[20] Ganju RK, Brubaker SA, Meyer J, et al. The alpha-chemokine, stromal cell-derivedfactor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4receptor andactivates multiple signal transduction pathways. J Biol Chem.1998;273:23169–23175.
[21] Dimmeler S, Aicher A, Vasa M, et al. HMG-CoA reductase inhibitors (statins)increase endothelial progenitor cells via the PI3-kinase/Akt pathway. J Clin invest.2001;108(3):391-397.
[22] Imanishi T, Moriwaki C, Hano T, et al. Estrogen reduces endothelial progenitor cellsenescence through augmentation of telomerase activity. J Hypertens.200523(9):1699–1706.
[23] Bahlmann FH, DeGroot K, Duckert T, et al. Endothelial progenitor cell proliferationand differentiation is regulated by erythropoietin. Kidney Int.2003;64:1648–1652.
[24] Wassmann S, Werner N, Czech T, et al. Improvement of endothelial function bysystemic transfusion of vascular progenitor cells. Circ Res.2006;99: e74–e83.
[1] Mangge H，Almer G, Truschnig-Wilders M, et al. Inflammation, adiponection,obesityand cardiovascular risk.Curr Med Chem.2010;17（36）:4511-4520
[2] LusisAJ.Atherosclerosis.Nature,2000,407(6801):233-241
[3] Rauscher FM, Goldchmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cellexhaustion, and atherosclerosis. Circulation.2003;108(4):457-463.
[4] Reyes M, Dudek A, Jahagirdar B, et al Origin of endothelial progenitors in humanpostnatal bone marrow. J Clin Invest.2002;109(3):337-346.
[5] Yin A H, Miraglia S, Zanjani ED, et al. AC133, a novel marker for humanhematopoietic stem and progenitor cells. Blood.1997,90(12):5002–5012.
[6] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelialcells for angiogenesis. Science.1997;(5302):964-967.
[7] Krause D S, Fackler MJ, Civin C I, et al.CD34: structure, biology, and clinical utility.Blood.1996;87(1):1–13.
[8] Karkkainen, M. J. and Petrova, T. V. Vascular endothelial growth factor receptors inthe regulation of angiogenesis and lymphangiogenesis. Oncogene,2000;19(49):5598–5605.
[9] Nowak G, Karrar A, Holmen C, et al. Expression of vascular endothelial growth factorreceptor-2or Tie-2on peripheral blood cells defines functionally competent cellpopulations capable of reendothelialization.Circulation.2004;110(24):3699–3707.
[10] Briasoulis A,Tousoulis D, Antoniades C, et al.The Role of Endothelial ProgenitorCells in Vascular Repair after Arterial Injury and Atherosclerotic PlaqueDevelopment. Cardiovascular Therapeutics.2010;29(20):125-139.
[11] Suda T, Takakura N, and Oike Y. Hematopoiesis and angiogenesis. Int J Hematol.2000;71(2):99-107.
[12] Fischer C, Schneider M, Carmeliet P. Principles and therapeutic implications ofangiogenesis, vasculogenesis and arteriogenesis. Handb Exp Pharmacol.2006;(176Pt2):157-212.
[13] Nakul-Aquaronne D, Bayle J, Frelin C. Coexpression of endothelial markers andCD14by cytokine mobilized CD34+cells under angiogenic stimulation. CardiovascRes.2003;57(3):816-823．
[14] Bonello L, Basire A, Sabatier F, et al. Endothelial injury induced by coronaryangioplasty triggers mobilization of endothelial progenitor cells in patients with stablecoronary artery disease. Thromb Haemost.2006;4(5):979-981.
[15] Harrison JS, Rameshwar P, Chang V, et al. Oxygen saturation in the bone marrow ofhealthy volunteers. Blood.2002;99(1):394.
[16] Ceradini, D J, Kulkarni, A R, Callaghan, M J. Progenitor cell trafficking is regulatedby hypoxic gradients through HIF-1induction of SDF-1. Nat. Med.2004;10(8):858-864.
[17] Semenza, G. Signal transduction to hypoxia-inducible factor1. Biochem. Pharmacol.2002;64(5-6):993–998.
[18] Heeschen C, Aicher A, Lehmann R., et al. Erythropoietin is a potent physiologicstimulus for endothelial progenitor cell mobilization. Blood.2003;102(4):1340–1346.
[19] Bahlmann FH, de Groot K, Spandau JM, et al. Erythropoietin regulates endothelialprogenitor cells. Blood.2004;103(3):921–926.
[20] Iwakura A, Luedemann, C, Shastry S, et al. Estrogen-mediated, endothelial nitricoxide synthase-dependent mobilization of bone marrow-derived endothelialprogenitor cells contributes to reendothelialization after arterial injury. Circulation.2003;108(25):3115–3121.
[21] Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitricoxide from l-arginine. Nature.1988;333(6174):664–666.
[22] Heissig B, Hattori K, Dias S, et al. Recruitment of stem and progenitor cells from thebone marrow niche requires MMP-9mediated release of kit-ligand. Cell.2002;109(5):625–637.
[23] Guthrie SM, Curtis LM, Mames RN, et al. The nitric oxide pathway modulateshemangioblast activity of adult hematopoietic stem cells. Blood.2005;105(5):1916–1922.
[24] Asahara, T, Takahashi, T, Masuda, H, et al. VEGF contributes to postnatalneovascularization by mobilizing bone marrow-derived endothelial progenitor cells.EMBO J.1999;18(14):3964-3972.
[25] Awad O, Dedkov E I, Jiao C. Differential healing activities of CD34+and CD14+endothelial cell progenitors. Arterioscler Thromb Vasc Biol.2006;26(4):758-64.
[26] Miller-Kasprzak E, Jagodzinski P.P. Endothelia progenitor cells as anew agentcontributing to vascular repair．Arch Immunol Ther Exp(Warsz).2007;55(4):247-259.
[27] Libby P. Inflammation in atherosclerosis. Nature.2002;420(6917):868-74.
[28] Fadini GP, Coracina A, Baesso I, et al. Peripheral blood CD34+KDR+endothelialprogenitor cells are determinants of subclinical atherosclerosis in a middle-agedgeneral population. Stroke.2006;37(9):2277-82.
[29] Thum T, Tsikas D, Stein S, et al. Suppression of endothelial progenitor cells in humancoronary artery disease by the endogenous nitric oxide synthase inhibitor asymmetricdimethylarginine. J. Am. Coll. Cardiol.2005;46(9):1693–1701.
[30] Chironi G, Walch L, Pernollet MG, et al. Decreased number of circulatingCD34+KDR+cells in asymptomatic subjects with preclinical atherosclerosis.Atherosclerosis.2007;191(1):115-120.
[31] Lau K K, Chan YH, Yiu KH, et al. Burden of carotid atherosclerosis in patients withstroke: relationships with circulating endothelial progenitor cells andhypertension. J. Hum. Hypertens.2007;21(6):445–451.
[32] Fadini GP, Sartore S, Albiero M, et al. Number and function of endothelial progenitorcells as a marker of severity for diabetic vasculopathy. Arterioscler Thromb VascBiol.2006;26(9):2140-6.
[33] Guven H, Shepherd R M, Bach RG, et al. The number of endothelial progenitor cellcolonies in the blood is increased in patients with angiographically significantcoronary artery disease. J. Am. Coll. Cardiol.2006;48(8):1579–1587.
[34] Leor J, and Marber M. Endothelial progenitors: a new Tower of Babel? J. Am. Coll.Cardiol.2006;48(8):1588–1590.
[35] Thum T, Bauersacha J. Mobilization of bone marrow-derived stem cells aftermyocardial infarction and left ventricular function: simply effects of optimized drugtreatment?. Eur.Heart J.2005;26(12):1196–1204.
[36] Lambiase PD, Edwards R J，Anthopoulos P, et al. Circulating humoral factors andendothelial progenitor cells in patients with differing coronary collateral support.Circulation.2004;109(24):2986-92.
[37] Werner N, Kosiol S，Schiegl T, et al. Circulating endothelial progenitor cells andcardiovascular outcomes. N Engl J Med.2005;353(10):999-1007.
[38] Rosenzweig A, Circulating endothelial progenitors--cells as biomarkers. N Engl JMed.2005;353(10):1055-7.
[39] Schmidt-Lucke C, et al. Reduced number of circulating endothelial progenitor cellspredicts future cardiovascular events: proof of concept for the clinical importance ofendogenous vascular repair. Circulation.2005;111(22):2981-2987.
[40] Kunz GA, Liang G, Cuculi F, et al. Circulating endothelial progenitor cells predictcoronary artery disease severity. Am Heart J.2006;152(1):190-195.
[41] Fadini, GP, de Kreutzenberg SV,Coracina A, et al. Circulating CD34+cells,metabolic syndrome, and cardiovascular risk. Eur Heart J.2006;27(18):2247-2255.
[42] Fadini GP, Agostini C,Boscaro E, et al. Mechanisms and significance of progenitorcell reduction in the metabolic syndrome. Metab Syndr Relat Disord.2009;7(1):5-10.
[43] Werner N, Wassmann S, Ahlers P, et al. Circulating CD31+/annexin V+apoptoticmicroparticles correlate with coronary endothelial function in patients with coronaryartery disease. Arterioscler. Thromb. Vasc. Biol.2006;26(1):112–116.
[44] Griese, D P, Ehsan, A, Melo, L G. Isolation and transplantation of autologouscirculating endothelial cells into denuded vessels and prosthetic grafts: implicationsfor cell-based vascular therapy. Circulation.2003;108(21):2710-2715.
[45] Takamiya M, Okigaki M, Jin D, et al. Granulocyte colony-stimulating factor-mobilized circulating Kit+/Flk-1+progenitor cells regenerate endotheliumand inhibitneointimal hyperplasia after vascular injury.Arterioscler Thromb Vasc Biol.2006;26(4):751–757.
[46] Foteinos G, Hu Y, Xiao Q, et al. Rapid endothelial turnover in atherosclerosis-proneareas coincides with stem cell repair in apolipoprotein E-deficient mice. Circulation.2008;117(14):1856–1863.
[47] Landmesser U, Engberding N, Bahlmann, FH, et al. Statin-induced improvement ofendothelial progenitor cell mobilization, myocardial neovascularization, leftventricular function and survival after experimental myocardial infarction requiresendothelial nitric oxide synthase. Circulation.2004;110(14):1933–1939.
[48] Wilkinson IB, Qasem A, McEniery CM., et al. Nitric oxide regulates local arterialdistensibility in vivo. Circulation.2002;105(2):213–217.
[49] Celermajer DS, Sorensen KE, Gooch VM, et al. Non-invasive detection of endothelialdysfunction in children and adults at risk of atherosclerosis. Lancet.1992;340(8828):1111–1115.
[50] Murphy C, Kanaganayagam GS, Jiang BC, et al.Vascular dysfunction and reducedcirculating endothelial progenitor cells in young healthy UK South Asian men.Arterioscler. Thromb. Vasc. Biol.2007;27(4):936–942.
[51] Wassmann S, Werner N, Czech T, et al. Improvement of endothelial function bysystemic transfusion of vascular progenitor cells. Circ. Res.2006;99(8): e74–e83.
[52] Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genesencoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivoarteriogenesis through paracrine mechanisms. Circ Res.2004;94(5):678–685.
[53] Cho HJ, Lee N, Lee JY, et al. Role of host tissues for sustained humoral effects afterendothelial progenitor cell transplantation into the ischemic heart. JExpMed.2007;204(13):3257–3269.
[54] Pirro M, Kazakov A,Semenov A, et al. Reduced number of circulating endothelialprogenitors and HOXA9expression in CD34+cells of hypertensive patients.JHypertens.2007;25(10):2093-9.
[55] Dong L, Kang L,Ding L,et al. Insulin modulates ischemia-induced endothelialprogenitor cell mobilization and neovascularization in diabetic mice. Microvasc Res.2011;82(3):227-36.
[56] Hibbert B, Ma X, Pourdjabbar A, et al. Pre-procedural atorvastatin mobilizesendothelial progenitor cells: clues to the salutary effects of statins on healing ofstented human arteries. PLoS One.2011;6(1): e16413.
[57] Hamada H, Kim M K, Iwakura A, et al. Estrogen receptors alpha and beta mediatecontribution of bone marrow-derived endothelial progenitor cells to functionalrecovery after myocardial infarction. Circulation.2006;114(21):2261-2270.
[58] Iwakura A, Shastry S, Luedemann C, et al. Estradiol enhances recovery aftermyocardial infarction by augmenting incorporation of bone marrow-derivedendothelial progenitor cells into sites of ischemia-induced neovascularization viaendothelial nitric oxide synthase-mediated activation of matrix metalloproteinase-9.Circulation.2006;113(12):1605–1614.
[59] Silva JF, Rocha NG, Nobrega AC. Mobilization of endothelial progenitor cells withexercise in healthy individuals: a systematic review. Arq Bras Cardiol.2012;98(2):182-191.
[60] Engelmann MG, Theiss HD, Hennig-TheissC,et al.Autologous bone marrow stem cellmobilization induced by granulocyte colony-stimulating factor after subacuteST-segment elevation myocardial infarction undergoing late revascularization: finalresults from the G-CSF-STEMI (Granulocyte Colony-Stimulating Factor ST-SegmentElevation Myocardial Infarction) trial. J. Am.Coll. Cardiol.2006;48(8):1712–1721.
[61] Powell TM, Paul JD, Hill JM, et al.Granulocyte colony-stimulating factor mobilizesfunctional endothelial progenitor cells in patients with coronary artery disease.Arterioscler. Thromb. Vasc. Biol.2005;25(2):296–301.
[62] Lipsic E, Van Der Meer P, Voors AA, et al. A single bolus of a long-actingerythropoietin analogue darbepoetin α in patients with acute myocardial infarction: arandomized feasibility and safety study. Cardiovasc. Drugs Ther.2006;20(2):135–141.
[63] Werner C, Kamani CH, Gensch C,et al.The peroxisome proliferator-activatedreceptor-γ agonist pioglitazone increases number and function of endothelialprogenitor cells in patients with coronary artery disease and normal glucose tolerance.Diabetes.2007;56(10):2609–2615.
[64] Cherqui S, Kurian S, M.Schussler O, et al. Isolation and angiogenesis by endothelialprogenitors in the fetal liver.Stem Cells.2006;24(1):44-54.
[65] Alessandri G, Girelli M, et al.Human vasculogenesis ex vivo: embryonal aorta as atool for isolation of endothelial cell progenitors. Lab Invest.2001;81(6):875-885.
[66] Invernici G,Emanueli C,Madeddu P, et al. Human fetal aorta contains vascularprogenitor cells capable of inducing vasculogenesis, angiogenesis, and myogenesis invitro and in a murine model of peripheral ischemia.Am J Pathol.2007;170(6):1879-1892.
[67] Furuhata S, Ando K, Oki M, et al. Gene expression profiles of endothelial progenitorcells by oligonucleotide microarray analysis. Mol Cell Biochem. Mol CellBiochem.2007;298(1-2):125-138.
[68] Miranville A, Heeschen C, Sengenes C, et al. Improvement of postnatalneovascularization by human adipose tissue-derived stem cells.Circulation.2004;110(3):349-55.