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.     

鼻咽癌患者外周血循环内皮细胞的检测及临床意义分析

目的分析鼻咽癌患者外周血循环内皮细胞（CECs）的水平及临床意义。 方法选取2016年1月至2018年10月期间于陆军军医大学第二附属医院接受单纯放疗或同期放化疗的55名初诊鼻咽癌患者为研究对象,归为鼻咽癌组,并随机选取同期来医院进行体检的50例健康成人为对照组。比较两组外周血CECs水平,并分析鼻咽癌组不同临床病理资料患者的外周血CECs水平,以及治疗前后的外周血CECs水平变化。根据疗效分为完全缓解（CR）组和未完全缓解组（包括部分缓解、疾病稳定和疾病进展）。两组间比较采用两样本t检验;计量资料采用百分比表示,比较采用χ²检验。 结果鼻咽癌组患者治疗前的外周血CECs水平为（21.13±8.33）个/μl,高于对照组的（5.03±2.25）个/μl,差异有统计学意义（t = 13.230,P < 0.01）。鼻咽癌组治疗前的外周血CECs水平T3~T4期（23.23±8.09）?个/μl高于T1~T2期（16.01±5.22）个/μl,差异具有统计学意义（t = 3.290,P < 0.01）;N1~N3期（22.82±8.16）?个/μl高于N0期（15.06± 3.98）个/μl,差异具有统计学意义（t = 3.176,P < 0.01）;M1期（28.30±3.33）?个/μl高于M0期（19.91±8.23）?个/μl,差异具有统计学意义（t = 2.826,P < 0.01）;Ⅲ~Ⅳ期（23.26±7.93）个/μl高于Ⅰ~Ⅱ期（17.93±5.63）?个/μl,差异具有统计学意义（t = 2.726,P < 0.01）。CR组患者治疗前（20.03±8.12）?个/μl、治疗后3个月（12.61±5.33）?个/μl的外周血CECs水平低于未完全缓解组（26.75±3.29）?个/μl、（19.03±2.62）?个/μl,差异具有统计学意义（t = 5.181、5.507,P均< 0.01）。 结论鼻咽癌患者的外周血CECs水平明显升高,与病情进展、放化疗效果有关,可能成为潜在的肿瘤标志物。     

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.     

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.     

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.     

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.     

Infarcted myocardium-like stiffness contributes to endothelial progenitor lineage commitment of bone marrow mononuclear cells

Optimal timing of cell therapy for myocardial infarction (MI) appears during 5 to 14 days after the infarction. However, the potential mechanism requires further investigation. This work aimed to verify the hypothesis that myocardial stiffness within a propitious time frame might provide a most beneficial physical condition for cell lineage specification in favour of cardiac repair. Serum vascular endothelial growth factor (VEGF) levels and myocardial stiffness of MI mice were consecutively detected. Isolated bone marrow mononuclear cells (BMMNCs) were injected into infarction zone at distinct time-points and cardiac function were measured 2 months after infarction. Polyacrylamide gel substrates with varied stiffness were used to mechanically mimic the infarcted myocardium. BMMNCs were plated on the flexible culture substrates under different concentrations of VEGF. Endothelial progenitor lineage commitment of BMMNCs was verified by immunofluorescent technique and flow cytometry. Our results demonstrated that the optimal timing in terms of improvement of cardiac function occurred during 7 to 14 days after MI, which was consistent with maximized capillary density at this time domains, but not with peak VEGF concentration. Percentage of double-positive cells for DiI-labelled acetylated low-density lipoprotein uptake and fluorescein isothiocyanate (FITC)-UEA-1 (ulex europaeus agglutinin I lectin) binding had no significant differences among the tissue-like stiffness in high concentration VEGF. With the decrease of VEGF concentration, the benefit of 42 kPa stiffness, corresponding to infarcted myocardium at days 7 to 14, gradually occurred and peaked when it was removed from culture medium. Likewise, combined expressions of VEGFR2(+) , CD133(+) and CD45(-) remained the highest level on 42 kPa substrate in conditions of lower concentration VEGF. In conclusion, the optimal efficacy of BMMNCs therapy at 7 to 14 days after MI might result from non-VEGF dependent angiogenesis, and myocardial stiffness at this time domains was more suitable for endothelial progenitor lineage specification of BMMNCs. The results here highlight the need for greater attention to mechanical microenvironments in cell culture and cell therapy.     

Translating preclinical findings of (endothelial) progenitor cell mobilization into the clinic; from bedside to bench and back

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.     

Improved angiogenic activity of endothelial progenitor cell in diabetic patients treated with insulin plus metformin

Type 2 diabetes (T2DM) is associated with an increased vascular disease. Moreover, endothelial progenitor cell (EPC) function is impaired in diabetic patients. Decreased EPC number plays a critical role in reduced endothelial repair and development of the vascular disorder. To determine the effect of metformin and insulin plus metformin on functional activity of EPCs, 130 participants were divided into three groups (group 1: healthy control; group 2: metformin; group 3: insulin plus metformin). The concentration of EPCs in the circulation was first quantified. Thereafter, circulating EPCs (cEPCs) were harvested and the biological features of these cells including proliferative, clonogenicity, tubulogenic, and migratory properties were analyzed after expansion. The serum protein levels of some proangiogenic factors were also measured. Our results showed greater numbers of cEPCs in control and in diabetic patients treated with insulin plus metformin than in metformin-treated patients. Insulin plus metformin therapy was associated with augmented proliferative, clonogenicity, migratory, and tubulogenic activity of cEPCs in patients with T2DM. Increased serum concentrations of angiogenic factors were also observed in patients treated with insulin plus metformin. Western blot analysis showed increased protein levels of pTie-2/Tie2 and Pakt/AKT in cEPCs harvested from T2DM, treated with insulin metformin plus. This study showed that treatment with insulin plus metformin in diabetic patients is associated with increased mobilization of EPCs into the circulation, with potential beneficial effect in vascular protection in diabetic patients.     

Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells

Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34(+) cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs.     

Identification and clinical significance of circulating endothelial progenitor cells in gastric cancer

Abstract

Context: 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.     

Identification of circulating CD90 CD73 cells in cirrhosis of liver

AIM:To identify circulating CD90 + CD73 + CD45 cells and evaluate their in vitro proliferating abilities.METHODS:Patients with cirrhosis(n=43),and healthy volunteers(n=40)were recruited to the study.Mononuclear cells were isolated and cultured from the peripheral blood of controls and cirrhosis patients.Fibroblast-like cells that appeared in cultures were analyzed for morphological features,enumerated by flow cytometry and confirmed by immunocytochemistry(ICC).Colony forming efficiency(CFE)of these cells was assessed and expressed as a percentage.RESULTS:In comparison to healthy volunteers,cells obtained from cirrhotic patients showed a significantincrease(P<0.001)in the percentage of CD90+CD73+ CD45 cells in culture.Cultured cells also showed 10 fold increases in CFE.Flow cytometry and ICC confirmed that the proliferating cells expressed CD90 + CD73 + in the cultures from cirrhosis patients.CONCLUSION:These results indicate the presence of circulating CD90 + CD73 + CD45 cells in patients with liver cirrhosis that have the potential to proliferate at a higher rate.     

Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co‐regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1‐MMP (MT1‐MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs.     

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro

Objective: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). Methods: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. Results: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. Conclusions: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.