Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells

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.     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

The involvement of endothelial progenitor cells in tumor angiogenesis

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood CD34, VEGFR-2, or AC 133 (CD133) antigen-positive cells, which may home to site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis and growth of certain tumors. This review article will summarize the literature data concerning this new role played by EPCs in tumor angiogenesis.     

他汀类药物对外周血内皮祖细胞的影响

本文旨在探讨他汀类药物氟伐他汀对外周血内皮祖细胞(endothelial progenitor cells,EPCs)数量和功能的影响.用密度梯度离心从外周血获取单个核细胞,将其接种在人纤维连接蛋白(human fibronectin)包被的培养板中,培养7 d后,收集贴壁细胞,加入不同浓度氟伐他汀(分别为0.01、0.1、1、10μmol/L)和辛伐他汀(1 μmol/L),培养一定的时间(6、12、24、48 h).用激光共聚焦显微镜鉴定FITC-UEA-I和DiI-acLDL双染色阳性细胞为正在分化的EPCs,用流式细胞仪检测其表面标志进一步鉴定EPCs,在倒置荧光显微镜下计数.采用MTT比色法、改良的Boyden小室、粘附能力测定实验和体外血管生成试剂盒观察EPCs的增殖能力、迁移能力、粘附能力和体外血管生成能力.结果显示,氟伐他汀可显著增加外周血EPCs的数量,并且EPCs数量随氟伐他汀浓度增加及作用时间延长而增加,1 μmol/L浓度氟伐他汀作用24h对EPCs的数量影响最为显著(较对照组增加15倍,P＜0.05).在动物实验中,喂养氟伐他汀3周后,大鼠的EPCs也较对照组增加2倍(P＜0.05),进一步支持了体外实验的结果.氟伐他汀和辛伐他汀也显著改善外周血EPCs的粘附能力、迁移能力、增殖能力和体外血管生成的能力,相同浓度的氟伐他汀和辛伐他汀(1 μmol/L)对EPCs数量和功能的影响并无显著差异.上述观察结果提示他汀类药物可增加EPCs的数量,改善EPCs功能.     

Role of aldehyde dehydrogenase 2 Glu504lys polymorphism in acute coronary syndrome

This study aimed to investigate the association of the aldehyde dehydrogenase 2 (ALDH2) Glu504Lys polymorphism, which exists in 30-50% of East Asians, and risk of acute coronary syndrome (ACS). We enrolled 1092 unrelated Han Chinese, including 546 with ACS and 546 age- and sex-matched controls. Subjects with ALDH2 mutant genotypes showed significantly higher ACS than did controls (46.7% versus 31.9%, P < 0.001). Logistic regression analysis revealed the ALDH2 mutant independently associated with ACS (odds ratio [OR] 1.95, 95% confidence interval [CI]: 1.31-2.92, P = 0.001), but the association was weaker on adjusting for alcohol consumption (OR 1.82, 95% CI: 1.23-2.70, P = 0.003). Similar results were found in a subgroup analysis of patients with primary ST-segment elevation myocardial infarction (STEMI). The ALDH2 mutant was significantly associated with level of high-sensitivity C-reactive protein (hs-CRP) in patients with ACS (P = 0.002) and in controls (P = 0.009) and number of circulating endothelial progenitor cells (EPCs) (P = 0.032); furthermore, inclusion of hs-CRP level and EPCs number as independent variables in regression analysis reduced the importance of ALDH2 polymorphism in ACS or primary STEMI. However, ALDH2 polymorphism was not associated with number of coronary arteries with significant stenosis, Gensini score or flow-mediated dilation of the brachial artery. Our results suggest that ALDH2 mutation is a genetic risk marker for ACS, which is explained in part by alcohol consumption, inflammation and number of circulating EPCs.     

Three specific antigens to isolate endothelial progenitor cells from human liposuction material

Background aimsHuman endothelial progenitor cells (EPC) play an important role in regenerative medicine and contribute to neovascularization on vessel injury. They are usually enriched from peripheral blood, cord blood and bone marrow. In human fat tissue, EPC are rare and their isolation remains a challenge.MethodsFat tissue was prepared by collagenase digestion, and the expression of specific marker proteins was evaluated by flow cytometry in the stromal vascular fraction (SVF). For enrichment, magnetic cell sorting was performed with the use of CD133 microbeads and EPC were cultured until colonies appeared. A second purification was performed with CD34; additional isolation steps were performed with the use of a combination of CD34 and CD31 microbeads. Enriched cells were investigated by flow cytometry for the expression of endothelial specific markers, by Matrigel assay and by the uptake of acetylated low-density lipoprotein.ResultsThe expression pattern confirmed the heterogeneous nature of the SVF, with rare numbers of CD133+ detectable. EPC gained from the SVF by magnetic enrichment showed cobblestone morphology of outgrowth endothelial cells and expressed the specific markers CD31, CD144, vascular endothelial growth factor (VEGF)R2, CD146, CD73 and CD105. Functional integrity was confirmed by uptake of acetylated low-density lipoprotein and the formation of tube-like structures on Matrigel.ConclusionsRare EPC can be enriched from human fat tissue by magnetic cell sorting with the use of a combination of microbeads directed against CD133, an early EPC marker, CD34, a stem cell marker, and CD31, a typical marker for endothelial cells. In culture, they differentiate into EC and hence could have the potential to contribute to neovascularization in regenerative medicine.     

人胚胎干细胞向内皮祖细胞诱导分化及其应用研究进展

近年来,内皮细胞的应用价值不断提高,应用领域不断拓宽,但其来源有限,成为研究应用的主要障碍.胚胎干细胞在体外可分化为多种组织细胞系,有可能成为获取内皮细胞的另一来源.就人胚胎干细胞向内皮祖细胞分化、分离方法、相关分子机制及内皮祖细胞应用价值等进行阐述,以期能够引起更多的关注,推动其研究的进展.     

Effects of osteopontin on functional activity of late endothelial progenitor cells

The aim of this study is to investigate the effect of osteopontin (OPN) on functional activity of late endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin‐coated culture plates. Late EPCs were positive for both 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindocarbocyanine‐labeled acetylated low‐density lipoprotein (DiI‐acLDL) and fluorescein‐isothiocyanate‐conjugated Ulex europaeus agglutinin lectin (UEA‐1). Expression of von Willbrand factor (vWF) and kinase insert domain receptor (KDR) were detected by indirect immunofluorescence staining. Late EPCs of 3–5 passages were treated for 24 h with OPN (to make a series of final concentration: 0.005 µg/ml, 0.01 µg/ml, 0.05 µg/ml, 0.5 µg/ml, 2.5 µg/ml), or vehicle control. The proliferation, migration, and in vitro vasculogenesis activity of late EPCs were assayed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay and an in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin‐coated plates, and then adherent cells were counted. Incubation with OPN dose‐dependently inhibited the proliferative, adhesive, and in vitro vasculogenesis capacity and increased migratory activity of late EPCs. J. Cell. Biochem. 112: 1730–1736, 2011. © 2011 Wiley‐Liss, Inc.     

Purification and characterization of human neural stem and progenitor cells

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Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis

Abstract

Although endothelial progenitor cells (EPCs) have been used to promote revascularization after peripheral or myocardial ischemia, excess amounts of reactive oxygen species (ROS) are often involved in senescence and apoptosis of EPCs, thereby causing defective neovascularization and reduced or failed recovery. Here, we examined the cytoprotective effect of Ecklonia cava-derived antioxidant dieckol (DK) on oxidative stress-induced apoptosis in EPCs to improve EPC bioactivity for vessel repair. Although H₂O₂ (10 ^{? 3} M) increased the intracellular ROS level in EPCs, DK (10ug/ml) pretreatment suppressed the H₂O₂-induced ROS increase and drastically reduced the ratios of apoptotic cells. H₂O₂-induced ROS increased the phosphorylation of p38 MAPK and JNK; this was inhibited by DK pretreatment. H₂O₂ treatment increased the phosphorylation of NF-κB, which was blocked by pretreatment with SB 203580, a p38 MAPK inhibitor, or SP 600125, a JNK inhibitor. H₂O₂ decreased the cellular levels of Bcl-2 and c-IAPs, cellular inhibitors of apoptosis proteins, but increased caspase-3 activation. However, all these effects were inhibited by pretreatment with DK. Injection of DK-mixed EPCs (DK + EPCs) into myocardial ischemic sites in vivo induced cellular proliferation and survival of cells at the ischemic sites and, thereby, enhanced the secretion of angiogenic cytokines at the ischemic sites. These results show that DK + EPC exhibit markedly enhanced anti-apoptotic and antioxidative capabilities, unlike that shown by EPCs alone; thus, they contribute to improved repair of ischemic myocardial injury through cell survival and angiogenic cytokine production.     

Age‐related impairment of endothelial progenitor cell migration correlates with structural alterations of heparan sulfate proteoglycans

Aging poses one of the largest risk factors for the development of cardiovascular disease. The increased propensity toward vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells to contribute to vascular repair and regeneration. Although there is evidence to suggest that colony forming unit‐Hill cells and circulating angiogenic cells are subject to age‐associated changes that impair their function, the impact of aging on human outgrowth endothelial cell (OEC) function has been less studied. We demonstrate that OECs isolated from cord blood or peripheral blood samples from young and old individuals exhibit different characteristics in terms of their migratory capacity. In addition, age‐related structural changes were discovered in OEC heparan sulfate (HS), a glycocalyx component that is essential in many signalling pathways. An age‐associated decline in the migratory response of OECs toward a gradient of VEGF significantly correlated with a reduction in the relative percentage of the trisulfated disaccharide, 2‐O‐sulfated‐uronic acid, N, 6‐O‐sulfated‐glucosamine (UA[2S]‐GlcNS[6S]), within OEC cell surface HS polysaccharide chains. Furthermore, disruption of cell surface HS reduced the migratory response of peripheral blood‐derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Together these findings suggest that aging is associated with alterations in the fine structure of HS on the cell surface of OECs. Such changes may modulate the migration, homing, and engraftment capacity of these repair cells, thereby contributing to the progression of endothelial dysfunction and age‐related vascular pathologies.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Chondrogenesis of human periosteum-derived progenitor cells in atelocollagen

Periosteum-derived progenitor cells (PDPCs) could be differentiated into cartilage using atelocollagen as a carrier and in the presence of transforming growth factor-β3 (TGF-β3). Chondrogenesis was verified by RT-PCR and Western blotting. Expression of the type II collagen mRNA was found from the differentiated PDPCs in atelocollagen 3 weeks after chondrogenic induction. The chondrogenic potential of the PDPCs was also verified by histochemical staining for type II collagen protein. Increased production of glycosaminoglycan shows that the PDPCs in atelocollagen could differentiate into chondrocytes under a chondrogenic environment. PDPCs can therefore be used as a cell source for cell-based therapies targeted toward the articular cartilage of the knee.     

Cultivation and differentiation characteristics of human limbal progenitor cells

BACKGROUND: The aim of this study was to investigate whether limbal progenitor cells can be cultured, expanded and differentiated in vitro not only to enter corneal differentiation but also towards RPE (retinal pigment epithelium) characteristics. METHODS: A 3mm broad strip of human corneoscleral limbal tissue was digested enzymatically and cells were set into cell culture. Differentiation status and characteristics, proliferation and phagocytotic activity were assessed by immunocytochemical staining in combination with digital and confocal microscopy. RESULTS: Immunocytological analysis revealed expression of Nestin and p63 marker suggesting progenitor cell properties. Mitotic activity was demonstrated by BrdU (bromodesoxyuridine) uptake. Upon consecutive passages, corneal differentiation markers were predominantly expressed. Phagocytotic activity was demonstrated via uptake of FITC (fluorescein isothiocyanate) labelled latex beads. RPE markers Bestrophin and Cytokeratin 8/18 as well as glial marker GFAP and neuronal marker MAP with respective controls were negative indicating no differentiation towards characteristics of retinal pigment epithelium or neural and glial lineage. CONCLUSIONS: The results suggest that isolation and cultivation of proliferating and phagocytotic cells from the human corneal limbus was achieved which showed characteristics of both progenitor and differentiated corneal cells. No evidence was found for the hypothesis of spontaneous differentiation potential towards RPE lineage or neuronal characteristics, providing evidence of the inherent directional capacity of limbal progenitor cells.     

VEGF-A and PlGF-1 stimulate chemotactic migration of human mesenchymal progenitor cells

Vascular endothelial growth factor (VEGF) has been indicated to play a role during endochondral ossification by stimulation of blood vessel invasion into hypertrophic cartilage resulting in its replacement by trabecular bone. We could demonstrate a dose-dependent chemoattractive effect of VEGF-A and PlGF-1, but not VEGF-E or VEGF-C, on human mesenchymal progenitor cells. Quantitative realtime PCR revealed the expression of VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4), which markedly declined during osteogenic differentiation. In addition, expression of neuropilin-1 and -2 was detected by RT-PCR. In an in vitro kinase assay, we could demonstrate activation of VEGFR-1 and VEGFR-2 upon stimulation with specific ligands. These findings are consistent with the idea that the chemotactic effect of VEGF-A on MPC is mediated via VEGFR-1, and that VEGF-A and PlGF-1, have a functional role for recruitment of osteoprogenitor cells in the course of endochondral bone formation or remodeling.     

Coexpression of osteogenic and adipogenic differentiation markers in selected subpopulations of primary human mesenchymal progenitor cells

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age-related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP-activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator-activated receptor-gamma2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor-beta1 (TGF-beta1) and promoted by bone morphogenetic protein 2 (BMP-2). Osteogenic culture conditions including BMP-2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development.     

Rapamycin inhibits proliferation and differentiation of human endothelial progenitor cells in vitro

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.