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1、,Endothelial Progenitor CellsCharacterization and Role in Vascular Biology,Definition They are circulating,bone marrow-derived cells that are functionally and phenotypically distinct from mature endothelial cells They can differentiate into endothelial cells in vitro,as assessed by expression profil
2、es and functional characteristics They can contribute to in vivo vasculogenesis and/or vascular homeostasis,Endothelial progenitor cell,内皮祖细胞是能直接分化为血管内皮细胞的前体细胞,Embryonic EPC,胚外中胚层卵黄囊血岛 与造血干细胞存在共同的前体,Adult EPC,Bone Marrow(3%BM-MNC),Peripheral Blood(0.2%MNC),Origin and differentiation of endothelial p
3、rogenitor cells,Hematopoietic stem cells,CD133+,CD34+,CD45,CD14+,CD45+,CD45+,CD14+,CD45+,CD14+,CD133+,KDR+,CD34+,CD14+,CD133+,Endothelial markers+,Endothelial markers+,EPCs,EPCs,Myeloid Precursor,Monocytes,Macrophage,EPCs myeloid subtype,Mature EC,?,?,?,?,CD14lowCD34lowCD133-,Endothelial markers+,Me
4、senchymal stem cells,C-kit-,CD34-,Tissue-resident stem cells,C-kit+,EPCs Development,Molecular Mechanisms,Transcription factor SCL/Tal 是参与原血干细胞分化的基本的转录因子。A basic helix-loop-helix transcription factor 小鼠SCL基因无效突变的纯合子引起死亡 表现为卵黄囊毛细血管内皮形成障碍、早期造血不能发育,VEGFR and VEGF Receptor VEGFR-1(flt-1)VEGFR-2(flk-1/KD
5、R)VEGFR-3(flt-4)Ligand VEGF、VEGF-B,C,D,E,EPCs Development,VEGFR and VEGF,研究显示VEGFR2缺陷鼠在 E8.5E9.5时因缺乏 内皮和造血细胞而死亡。Flk1-/-的胚胎干细胞离体不能分化为EPC。胚胎干细胞向内皮细胞分化对VEGF呈剂量依赖性，VEGF浓度增加可增加原血干细胞向EPC转化，而 相应减少造血干细胞的生成。VEGFR1纯合突变鼠也因内皮细胞不能形成管样结 构而使胚胎在E9.5E10死亡。,VEGF是唯一已知的在胚胎杂合子状态致死的常染色体基因。,Tie receptor and ligand：受体酪氨酸激酶
6、家族 receptor Tie-1(Tie)Tie-2(Tek)Tie-2 ligand：Angiopoietin(Ang，血管生成素)Ang-1：血管内皮化 Ang-2：血管周围细胞和内皮细胞分离,EPCs Development,Tie receptor and ligand：受体酪氨酸激酶家族 Tie-2缺陷的胚胎不能建立血管结构的完整性 Ang-1缺陷鼠也表现为血管生成缺陷,EPCs Development,Ephs family Erythropoietin producing hepatocyte receptor(Eph)Ligand:ephrins(Eph A、B)Eph B可
7、调节 Ang-1 和 Tie-2的表达,EPCs Development,促红细胞生成素肝细胞受体及其配体，是酪氨酸激酶家族中的最大成员。基因缺失及体外血管形成实验表明：EphB和ephrinB在胚胎血管分化及成人病理性血管形成中发挥重要作用。,CD34+,Flk-1+,CD34+,Flk-1+,AC133+,CD34+,CD34+,Flk-1+,AC133-,Stem cell,EPC,HSC,EPC,EC,HSC,Circulating,EPC,EC,EPC,EC,CD34-selected culture-dish non-adherent putative EPC(CDNAC)chan
8、ge to a more mature endothelial phenotype during differentiation while the progenitor phenotype remains stable and the monocytic phenotype decreases.,CD34-selected putative EPC from culture-dish adherent cells(CDAC)express lower levels of endothelial and progenitor markers than CDNAC.,VEGFR-2/Flk-1+
9、,CD31+/Tie-2+,VE-Cadeherin+/Tie-1+,AC133+,AC133-,CD34+/VEGFR-2+cells can behave as EPCs.CD133+/CD34+/VEGFR-2+cells represent a more primitive EPC.,Putative EPCs take up acetylated low density lipoprotein(Ac-LDL)and bind the endothelial specific lectin UEA-1.,Mobilization of EPCs,Determined by local
10、microenvironment(stem cell niche),Fibroblasts,Osteoblasts,Endothelial cells,Stromal cells,Mobilization of EPCs,Stromal cells,Stem cells,Mobilizing cytokinesVEGF,SDF-1,G-CSF,EPOStatins,estrogen,exercise,hamper,Transendothelial migrationElastase,Cathepsin,MMPs,Blood circulating,Chemotaxis,Migration an
11、d Invasion SDF-1 VEGF,Adhesion Integrin Selectin,Differentiation VEGF,EPCs were originally thought to be present only during embryonic development.Evidence accumulating over several years suggests that they can persist in adult life.This has generated interest in the use of EPCs for neovascularizati
12、on of ischemic or injured tissue and for the clinical assessment of risk factors for various diseases.,EPC,Tumor angiogenesis,Ischemic vasculogenesis/Wound healing,Vascular homeostasis,EPCsfunction,Embryo vascular network expand Revascularization,Angiogenesis Vasculogenesis,Blood vessel development,
13、血管发生/生成,血管生成/新生/增生,不但是正常生理变化中(如生长、伤口愈合)所必需有的过程，和肿瘤的发展也有密切的关系。,Angiogenesis a regulated process involving the proliferation,and migration of endothelial cells from adjacent pre-existing blood vessels.,血管生成,指由已经存在的血管成熟内皮细胞通过局部芽生或套叠的形式生成新的毛细血管，是血管从少到多的过程，是血管新生的经典理论。,Vasculogenesis a regulated process f
14、ollowing differentiation of endothelial progenitor cells from mesodermal precursors/endothelial progenitor cells(EPCs),血管发生,这种方式最早发生在胚胎期的血管生成，直接由EPCs或更早来源的血管母细胞（angioblast）长成，是血管从无到有的过程。最近证据表明，EPCs参与的血管发生亦参与了出生后机体局部缺血组织的修复。,Vasculogenesis和Angiogenesis是血管发育过程中的两个阶段，但在中文翻译时出现了意思重叠和相近、不便于区别的问题。,传 统Angi
15、ogenesis主要是EC迁移增殖参与的，vasculogenesis在胚胎期和生后均存在，主要依靠EPC分化增殖参与。现 在1997年，Angiogenesis概念被打破，Asahara等证实除有EC迁移增殖参与，还有EPC的参与。,Decrease of endothelial progenitor cells in the blood is a risk factor for vascular disease.Depletion or senescence of endothelial progenitor cells may contribute to blood vessel dis
16、ease.,Endothelial progenitor cell,EPCs and endothelial regeneration,In the past,endothelial regeneration has been attributed to the migration and proliferation of neighboring EC In 1997,Asahara first demonstrated EPC contributed to endothelial regeneration,EPC and Neovascularization,Transplantation
17、of genetically modified cell(Rosa-26,GFP,Lac Z),Basal incorporation rate:extremely low In ischemic tissue,the incorporation rate:up to 90%High rate was predominantly detected in models of tumor angiogenesis Therapeutic application of cells by infusion(iv)of purified BM mononuclear cells or expanded
18、EPCs led to a higher incorporation rate(7 20%)as compared with endogenously mobilized BM-engrafted cells(2%),Mechanisms by Which EPCs Improve Neovascularization,Incorporation of EPCs Release of proangiogenesis factors in paracrine manner VEGF HGF IGF-1,VEGF:Vascular endothelial growth factor HGF:hep
19、atocyte growth factor IGF:Insulin-like Growth Factor I,EPC culture assay,Bone marrow,Histopaque 1083,Methods,Mononuclear cells(MNCs)were isolated from mouse bone marrow with Histopaque 1083 by density gradient centrifugation.,Bone marrow,Histopaque 1083,Resuspend in EGM2 Plate 5X106 cells/well onfib
20、ronectin-coated 6-well dish,Methods,MNCs were cultured in EGM2 with the supplement of VEGF,b-FGF,IGF-1 on fibronectin-coated 6-well plate for 2 days to remove mature endothelial cells and monocytes.,Bone marrow,Histopaque 1083,Resuspend in EGM2 Plate 5X106 cells/well onfibronectin-coated 6-well dish
21、,Methods,The non-adherent cells(which contain EPCs)are then harvested and plated on fibronectin-coated 24-well plates for 3 days.,Bone marrow,Histopaque 1083,Resuspend in EGM2 Plate 5X106 cells/well onfibronectin-coated 6-well dish,2 days,Collect no-adherent cellsPlate 1X106 cells/well onfibronectin
22、-coated 24-well dish,CD31,vWF and eNOS,Methods,2/3 volume culture media was changed every 2 days.Immunofluorescence staining for CD31,vWF and eNOS were performed and were observed under fluromicroscope.,Peripheral Blood Mononuclear Cells at Day 1,Peripheral Blood Mononuclear Cells at Day 2,Day 5,UEA
23、-1,DiI-acLDL,vWF,CD31,Matrix gel,Matrix gel,SMA,F4/80,CD31/vWF,Immunofluorescence staining of CD34-selected putative EPC from CDNAC after 4 weeks of culture,一个新的肿瘤血管标志分子。该分子在新生血管中选择性高表达，参与血管生成过程。,Questions,Identification and origin of EPCs.Optimization of culture conditions to enhance the benefit of EPCs therapy?Signals for EPCs homing and differentiation?,
