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In vitro differentiation characteristics of cultured human mononuclear cells-implications for endothelial progenitor cell biology 总被引:9,自引:0,他引:9
Walenta K Friedrich EB Sehnert F Werner N Nickenig G 《Biochemical and biophysical research communications》2005,333(2):476-482
Endothelial progenitor cells (EPCs) have been implicated in the pathogenesis and treatment of cardiovascular disease. By use of quantitative uptake of DiLDL and lectin staining, EPCs have been characterized reliably. However, the exact nature and function of this cell population still remains poorly defined. In an attempt to further clarify the cell surface characteristics of EPCs, mononuclear cells (MNCs) were isolated from human blood and cell surface expression patterns were defined by FACS analysis before and after differentiation for 1-10 days in cell culture. "Classical" double staining for DiLDL and Ulex europaeus increases to 89.2 /- 0.05 after 10 days in culture. Looking at EPC-specific markers by FACS analysis, 0.18 +/- 0.11% of freshly isolated MNCs express CD34, 0.13 +/- 0.08% CD133, 0.59 +/-0.51% VEGFr2, 0.01 +/- 0.02% CD34/VEGFr2, 0.09 +/- 0.05% CD34/CD133, 0.58 +/- 0.13% CD34/CD31, and 0.02 +/- 0.01% CD34/CD146, respectively. Induction of the endothelial phenotype is evidenced by positive staining for VEGFr2, CD146, and CD31, and occurs in co-expression with stem cell markers in less than 2 +/- 0.52% of cultured cells. Expression of CD34 increases to 0.38 +/- 0.10% after 10 days, whereas the CD133(+) cell population shows an initial peak at 24h (0.29 +/- 0.18%) before decreasing to 0.15 +/- 0.02% at day 10. EPCs co-expressing CD34/CD133 increase to 0.19 +/- 0.09% after 10 days, and EPCs double-positive for CD34/VEGFr2 increase to 1.45 +/- 1.03%. Looking at leukocyte, lymphocyte, and monocyte lineage markers, 56.27 +/- 0.15% of freshly isolated MNCs express CD45, 7.13 +/- 0.02% CD14, and 38.65 +/- 0.01% CD3. Over the 10-day culture period, expression of CD45 decreases to 28.48 +/- 0.18%, CD3 to 23.11 +/- 0.02%, and CD14 to 0.09 +/- 0.02%. Cells co-expressing CD3/CD45 decrease from 38.88 +/- 0.33% to 24.86 +/- 2.49% after 10 days in culture. These findings extend present knowledge by showing that human MNCs differentiate at a very low rate to EPCs, while a majority of the cultured cell population remain committed to the leukocyte or lymphocyte lineage. Careful surface marker analysis might be necessary when using in vitro EPC differentiation systems. 相似文献
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Zhang W Zhang G Jin H Hu R 《Biochemical and biophysical research communications》2006,348(3):1018-1023
Evidence for dysfunction of endothelial repair in aged mice was sought by studying the pattern of induced differentiation, quantity, and function of bone marrow-derived endothelial progenitor cells (EPCs) in aged mice. The CD117-positive stem cell population was separated from bone marrow by magnetic activated cell-sorting system (MACS), and EPCs were defined by demonstrating the expression of CD117+CD34+Flk-1+ by flow cytometry. After 7 days of culture, the number of clones formed was counted, and proliferation and migration of EPCs were analyzed by MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and modified Boyden chamber assay. The results demonstrated that compared to the control group, the quantity of bone marrow-derived CD117+ stem cells and EPCs, as well as the proliferation, migration, the number of clones formed, and phagocytotic function of EPCs were significantly reduced in aged mice. There were no significant differences in the morphology and induced differentiation pattern of EPCs between the aged mouse group and the control group. Authors suggest that the dysfunction of EPCs may serve as a surrogate parameter of vascular function in old mice. 相似文献
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Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood 总被引:6,自引:0,他引:6
The hierarchy of endothelial progenitor cells (EPCs) in human umbilical cord blood has been disclosed. In this study we compare, for the first time, the angiogenic potential difference between two types of EPCs. We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell-conditioned medium and obtained two types of EPCs, referred to as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs). Both types of cells possess characteristics of EPCs, including expressing CD31, VE-cadherin, KDR and von Willebrand factor, uptake of Ac-LDL and binding to lectin. However, unlike CACs, which express CD14 but not CD133, HPP-EPCs express CD133 but not CD14. Also, unlike CACs, HPP-EPCs display stronger proliferation and clonogenic potential in vitro and show stronger ability to promote vascular growth in the hind-limb model of ischemia in mice (BALB/C-nu) in vivo. 相似文献
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内皮祖细胞对于维持血管内皮完整性和血管稳态具有重要作用.增强EPC的数量和功能可使心血管疾病患者获益.炎症、氧化应激对内皮祖细胞动员及其功能发挥具有重要影响,本文着重综述炎症和氧化应激对内皮祖细胞动员的调控,并探讨增进内皮祖细胞数量和功能的相关治疗策略. 相似文献
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Yang N Li D Jiao P Chen B Yao S Sang H Yang M Han J Zhang Y Qin S 《Cytotechnology》2011,63(3):217-226
Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find
favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation
capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction
times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were
studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth
supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with
10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies
were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate
and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow
cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for
both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression
levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments
suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly
proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary
for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have
the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported. 相似文献
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Shibata R Skurk C Ouchi N Galasso G Kondo K Ohashi T Shimano M Kihara S Murohara T Walsh K 《FEBS letters》2008,582(11):1607-1612
Obesity-linked diseases are associated with suppressed endothelial progenitor cell (EPC) function. Adiponectin is an adipose-derived protein that is downregulated in obese and diabetic subjects. Here, we investigated the effects of adiponectin on EPCs. EPC levels did not increase in adiponectin deficient (APN-KO) in response to hindlimb ischemia. Adenovirus-mediated delivery of adiponectin increased EPC levels in both WT and APN-KO mice. Incubation of human peripheral blood mononuclear cells with adiponectin led to an increase of the number of EPCs. Adiponectin induced EPC differentiation into network structures and served as a chemoattractant in EPC migration assays. These data suggest that hypoadiponectinemia may contribute to the depression of EPC levels that are observed in patients with obesity-related cardiovascular disorders. 相似文献
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Neumüller J Neumüller-Guber SE Lipovac M Mosgoeller W Vetterlein M Pavelka M Huber J 《Histochemistry and cell biology》2006,126(6):649-664
The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34+ hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2–3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation. 相似文献
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《Biomarkers》2013,18(6):487-492
AbstractContext: There are few reports of endothelial progenitor cells (EPCs) in peripheral blood have been found in patients with gastric cancer.Objective: We quantified EPCs in the peripheral blood of patients with gastric cancer, with the expectation that this approach might lead to a new marker for the diagnosis of gastric cancer.Methods: We enumerated CD34+/CD133+ EPCs in the peripheral blood of 145 subjects by use of flow cytometry.Results and conclusion: The quantity of peripheral blood EPCs in patients with gastric cancer are correlated with patient’s age. In addition, the number of peripheral blood EPCs in patients with gastric cancer increased with tumor node metastasis stage and histological differentiation of the cancers, and with the operative status of the patients. 相似文献
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Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes. 相似文献
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Effects of transplanted circulating endothelial progenitor cells and platelet microparticles in atherosclerosis development 下载免费PDF全文
Adriana Georgescu Nicoleta Alexandru Eugen Andrei Emanuel Dragan Daniel Cochior Sérgio Dias 《Biology of the cell / under the auspices of the European Cell Biology Organization》2016,108(8):219-243
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Asakage M Tsuno NH Kitayama J Kawai K Okaji Y Yazawa K Kaisaki S Osada T Watanabe T Takahashi K Nagawa H 《Cancer immunology, immunotherapy : CII》2006,55(6):708-716
Endothelial progenitor cells (EPCs) have been recently found to exist circulating in peripheral blood of adults, and home
to sites of neovascularization in peripheral tissues. They can also be differentiated from peripheral blood mononuclear cells
(PBMNCs). In tumor tissues, EPCs are found in highly vascularized lesions. Few reports exist in the literature concerning
the characteristics of EPCs, especially related to their surface antigen expressions, except for endothelial markers. Here,
we aimed to investigate the surface expression of differentiation markers, and the functional activities of early-outgrowth
of EPCs (EO-EPCs), especially focusing on their antigen-presenting ability. EO-EPCs were generated from PBMNCs, by culture
in the presence of angiogenic factors. These EO-EPCs had the morphological and functional features of endothelial cells and,
additionally, they shared antigen-presenting ability. They induced the proliferation of allogeneic lymphocytes in a mixed-lymphocyte
reaction, and could generate cytotoxic lymphocytes, with the ability to lyze tumor cells in an antigen-specific manner. The
antigen-presenting ability of EO-EPCs, however, was weaker than that of monocyte-derived dendritic cells, but stronger than
peripheral blood monocytes. Since EO-EPCs play an important role in the development of tumor angiogenesis, targeting EPCs
would be an effective anti-angiogenic strategy. Alternatively, due to their antigen-presenting ability, EO-EPCs can be used
as the effectors of anti-tumor immunotherapy. Since they share endothelial antigens, the activation of a cellular immunity
against angiogenic vessels can be expected. In conclusion, EO-EPCs should be an interesting alternative for the development
of new therapeutic strategies to combat cancer, either as the effectors or as the targets of cancer immunotherapy. 相似文献
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Rapamycin inhibits proliferation and differentiation of human endothelial progenitor cells in vitro 总被引:16,自引:0,他引:16
Butzal M Loges S Schweizer M Fischer U Gehling UM Hossfeld DK Fiedler W 《Experimental cell research》2004,300(1):65-71
Bone-marrow-derived, circulating endothelial precursor cells contribute to neoangiogenesis in various diseases. Rapamycin has recently been shown to have anti-angiogenic effects in an experimental tumor model. Our group has developed a culture system that allows expansion and endothelial differentiation of human CD133(+) precursor cells. We could show by PCR analysis that mTOR, the rapamycin-binding protein, was expressed in fresh CD133(+) cells, in expanded cells after 28 days, and in differentiated endothelial cells. Rapamycin inhibited proliferation of CD133(+) cells dose dependently at similar concentrations as hematopoietic Jurkat or HL-60 cells. Apoptosis was induced by rapamycin after 48 h of treatment, which could be reduced by preincubation with FK 506. Furthermore, the development of adherent endothelial cells from expanded CD133(+) cells was dose dependently inhibited. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was reduced by rapamycin. In summary, rapamycin inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects. 相似文献
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Michele Moschetta Yuji Mishima Ilyas Sahin Salomon Manier Siobhan Glavey Angelo Vacca Aldo M. Roccaro Irene M. Ghobrial 《生物化学与生物物理学报:癌评论》2014
Tumor-associated neovasculature is a critical therapeutic target; however, despite significant progress made in the clinical efficacy of anti-vessel drugs, the effect of these agents remains transient: over time, most patients develop resistance, which inevitably leads to tumor progression. To develop more effective treatments, it is imperative that we better understand the mechanisms involved in tumor vessel formation, how they participate to the tumor progression and metastasis, and the best way to target them. 相似文献
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Interaction of platelets with endothelial progenitor cells in the experimental atherosclerosis: Role of transplanted endothelial progenitor cells and platelet microparticles 下载免费PDF全文
Nicoleta Alexandru Eugen Andrei Emanuel Dragan Adriana Georgescu 《Biology of the cell / under the auspices of the European Cell Biology Organization》2015,107(6):189-204
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Characterisation of the interaction between circulating and in vitro cultivated endothelial progenitor cells and the endothelial barrier 总被引:2,自引:0,他引:2
Funcke F Hoyer H Brenig F Steingen C Ladage D Müller-Ehmsen J Schmidt A Brixius K Bloch W 《European journal of cell biology》2008,87(2):81-90
In vitro cultured endothelial progenitor cells (cEPC) are used for intracoronary cell therapy in cardiac regeneration. The aim of this study was to investigate whether cEPC and circulating mononuclear cells (MNC), which include a small number of in vivo circulating EPC, are able to transmigrate through the endothelial barrier into the cardiac tissue. MNC and EPC were isolated from the peripheral blood from healthy male volunteers (n = 13, 25+/-6 years) and stained with a fluorescent marker. The cells were perfused in vitro through organs with endothelial layers of different phenotypes (rat aorta, human umbilical vein, isolated mouse heart). The endothelium and the basal lamina were then stained by immunofluorescence and the cryo-sections analysed using a confocal laser scanning microscope. After perfusion through the rat aorta, an adhesion/integration of MNC was observed at the endothelial layer and the basal lamina beneath endothelial cells. However, no migration of MNC over the endothelial barrier was found. This remained true even when the cell numbers were increased (from 0.5 to 10 million cells/h), when the time of perfusion was prolonged (1.5-4 h) and when the aorta was cultivated for 24 h. In the Langendorff-perfused mouse heart with intact endothelium, no migration of MNC (1 x 10(7)) or cEPC (1 x 10(6)) was observed after 0.5 and 2 h. In conclusion, MNC and cEPC do not possess any capacity to transmigrate the endothelial barrier. In the context of stem cell therapy, these cells may therefore serve as endothelial regenerators but not as cardiomyocyte substitutes. 相似文献
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Differentiation of circulating endothelial progenitor cells to a cardiomyogenic phenotype depends on E-cadherin 总被引:4,自引:0,他引:4
Koyanagi M Urbich C Chavakis E Hoffmann J Rupp S Badorff C Zeiher AM Starzinski-Powitz A Haendeler J Dimmeler S 《FEBS letters》2005,579(27):6060-6066
Progenitor cells may contribute to cardiac regeneration. Here, we investigated the role of cadherins and integrins for differentiation of human adult circulating endothelial progenitor cells (EPCs) into cardiomyocytes (CM) in a co-culture system. N- and E-cadherin were expressed in EPCs and were localized at the interface between EPCs and CM. Incubation of a blocking antibody against E-cadherin reduced the expression of CM marker protein in EPCs. Blocking antibodies against N- or P-cadherin or the beta1- and beta2-integrins were not effective. These data suggested that cell-to-cell communication mediated by E-cadherin contributes to the acquirement of a cardiomyogenic phenotype of human endothelial progenitor cells. 相似文献
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It is generally accepted that angiogenesis plays a major role in tumor growth and numerous targeting agents directed against angiogenesis pathways have been developed and approved for clinical use. In the past years the concept of angiogenesis has developed into a multi-faceted process in which, besides local activation and division of endothelial cells, bone marrow derived progenitor cells (BMDPCs) contribute to neovascularization. A multitude of preclinical and clinical data indicates that the release of BMDPCs influences the response to certain anti-cancer modalities. In this review we provide an overview of all the preclinical and clinical studies contributing to this hypothesis and translate these findings to the clinic by pointing out the clinical implications these findings might have. The recent insight in the mechanism of a systemic host response, in response to various treatment modalities has shed new light on the mechanism of tumor regrowth, early recurrence and metastasis formation during or after treatment. This provides various new targets for therapy which can be used to improve conventional chemotherapy. Furthermore it provides a potential explanation why bevacizumab selectively enhances the effectiveness of only certain types of chemotherapy. 相似文献
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Yu Y Gao Y Wang H Huang L Qin J Guo R Song M Yu S Chen J Cui B Gao P 《Experimental cell research》2008,314(17):3198-3208
Neovascularization and re-endothelialization relies on circulating endothelial progenitor cells (EPCs), but their recruitment and angiogenic roles are subjected to regulation by the vascular microenvironment, which remains largely unknown. The present study was designed to investigate the effects of mature ECs and matrix protein CCN1 on the properties of EPCs. In a coculture system, effects of ECs on proliferation, migration and participation in tube-like formation of EPCs were evaluated, and functional assays were employed to identify the exact role of CCN1 in EPCs vitality and function. We demonstrated that ECs, as an indispensable part of the cellular milieu, significantly promoted the proliferation, migration and tube formation activities of EPCs, and more importantly, CCN1 was potentially involved in such effects of ECs. Expression of CCN1 in EPCs was significantly increased by serum, VEGF, ECs-cocultivation and ECs conditioned medium. Moreover, Ad-CCN1-mediated overexpression of CCN1 directly enhanced migration and tube formation of EPCs, whereas silencing of endogenous CCN1 in EPCs inhibits cell functions. Furthermore, CCN1 induced the expressions of chemokines and growth factors, such as MCP-1 and VEGF, suggesting a complex interaction between those proangiogenic factors. Our data suggest that matrix protein CCN1 may play an important role in microenvironment-mediated biological properties of EPCs. 相似文献