内皮祖细胞生物学特性及其进展

内皮祖细胞(Endothelial Progenitor Cells,EPCs)是血管内皮细胞的前体细胞,即能分化为成熟血管内皮细胞的祖细胞。随着对EPCs功能和影响其分化、生存、归巢和组织分布因素的了解,EPCs作为临床诊断、预后判断和治疗方法将有广阔的前景。本文就EPCs的的来源,EPCs的分离、培养、鉴定,EPCs的表面标志,EPCs的动员、分化和归巢等生物学特性及其进展展开综述。     

内皮祖细胞研究进展

内皮细胞是能直接分化为血管内皮细胞的前体细胞,不仅参与胚胎血管生成,而且最近研究发现其也参与出生后的血管新生过程,提示内皮细胞在缺血性疾病、创伤愈合中的重要治疗作用和广阔的临床运用前景。本文将就目前有关内皮祖细胞的来源、发育分化、生理病理意义和应用研究作一综述。     

肝细胞生长因子对主要器官/组织损伤的修复作用

肝细胞生长因子(HGF)是一种多功能的生长因子,它参与多种细胞的增殖、迁移和形态发生。HGF对多种成熟的器官／组织有营养修复作用,促进肝、肾、肺等损伤器官的再生,同时也是神经系统新的营养因子之一。本综合近年来有关献对HGF在主要器官或组织损伤中的修复作用作一概括性介绍。     

粒细胞集落刺激因子动员内皮祖细胞预防再狭窄的研究

目的:观察粒细胞集落刺激因子是否能通过加速内皮修复预防再狭窄,并探讨该作用与其动员效应即EPCs数量和功能变化的相关性.方法:30只SD大鼠随机均分为假手术组、损伤未干预组和G-CSF组,后两组行颈总动脉球囊损造模,G-CSF组连续给药7天后观察各组外周血内皮祖细胞的数量和增值、粘附、迁移功能,4周后观察再狭窄和再内皮化程度.结果:G-CSF组再内皮化率高于对照组,再狭窄率低于对照组,再内皮化率和再狭窄率呈线性负相关;G-CSF组内皮祖细胞数量明显增加,内皮祖细胞增值、粘附、迁移功能也明显提高.结论:G-CSF通过加速内皮修复能预防再狭窄,该作用与其动员效应即内皮祖细胞数量的增加和增值、粘附、迁移功能的提高有关.     

微孔法分离大鼠骨髓内皮祖细胞

用一种杂交瘤皿,根据内皮祖细胞集落形成单位(endothelial progenitor cells colony-forming units,EPCs-CFUs)的形态特征和EPCs表面特异性标记物分离EPCs.取大鼠股骨、胫骨骨髓,将全骨髓接种在聚苯乙烯制作的杂交瘤皿上,培养4～7天后出现CFUs,将这些集落分别挑选出来后,取单个集落的部分细胞免疫荧光鉴定EPCs表面特异性标记物CD133/VEGFR-2.CD133/VEGFR-2双阳性即为EPCs-CFUs.与此对应的余下一部分继续传代增殖,流式细胞术鉴定CD133/VEGFR-2/CD34,并把此方法命名为微孔法.发现接种后第4天,显微镜下可见明显的CFUs.免疫荧光鉴定大约7%的CFUs为CD133 /VEGFR-2 ,进一步传代培养,流式细胞术鉴定CD133 /VEGFR-2 /CD34 细胞纯度达70%以上.传代细胞可在体外形成血管样结构,并表达内皮细胞特异性标记物vWF.结果表明通过微孔法能成功地从大鼠骨髓分离到EPCs.     

肝细胞生长因子

肝细胞生长因子吴青（中山大学生命科学学院,广州５１０２７５）关键词肝细胞生长因子１９８４年,ＭｉｃｈａＬｏｐｏｕｌｏｓ发现部分肝切除后的大鼠血浆中含有一种能刺激肝细胞ＤＮＡ合成的物质,１９８８年Ｇｏｈａｌａ从暴发性肝衰竭病人血浆中分离出类似物质。该类...     

子痫前期患者体内内皮祖细胞数量及VEGF表达水平变化研究

检测内皮祖细胞数量及VEGF表达水平在子痫前期患者和健康孕妇外周血循环间的差异,探究二者与子痫前期发病间的关系,为追溯子痫病病因和寻求子痫病发病的生物学指标提供依据。选取于本院就诊治疗的子痫前期患者和健康孕妇各30例,分别记为患病组和健康组。抽取外周静脉血,用密度梯度离心法分离单个核细胞,用CD34、CD133双荧光标记鉴定细胞,流式细胞仪检测细胞数量,酶联免疫吸附法(ELISA)检测血清VEGF表达水平。子痫前期患者外周静脉血中的内皮祖细胞数量明显少于健康孕妇组,VEGF表达水平也明显低于健康孕妇组,所有差异都具有统计学意义(p0.05)。子痫前期患者外周血循环中的内皮祖细胞数量明显减少,VEGF表达减少,这可能是子痫前期患者发病的原因之一。     

内皮祖细胞在炎症损伤修复中的作用和机制

内皮祖细胞（endothelial progenitor cells,EPCs）是出生后,可以在机体内分化为成熟内皮细胞的一种前体细胞,主要来源于骨髓。多种伴有血管内皮细胞损伤的疾病都可引起外周血EPCs数量变化。有研究显示EPCs参与炎性损伤修复,并且外周血EPCs数量与血管内皮损伤程度和疾病预后存在一定的相关关系。EPCs。通过动员、迁移、归巢和分化等步骤修复内皮。炎症反应中受损组织释放的基质细胞衍生因子、血管内皮生长因子可与EPCs相应的受体结合,通过内皮型一氧化氮合酶、基质金属蛋白酶9等途径调节内皮修复过程,这是EPCs分化为内皮细胞过程的主要调控机制。此外,EPCs还可通过旁分泌机制促进相邻的内皮细胞增殖分化。目前,EPCs在炎症领域仅用于内皮炎性损伤和疾病预后评估,但是EPCs在心血管疾病和组织工程领域应用研究的成功,为EPCs在炎症反应的诊断和治疗提供了新的思路。     

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.     

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.     

Hepatocyte growth factor induces angiogenesis in injured lungs through mobilizing endothelial progenitor cells

Circulating endothelial progenitor cells (EPCs) play a pivotal role in angiogenesis. Hepatocyte growth factor (HGF) is known to induce proliferation and motility in endothelial cells, and to play a role in mitogenic and morphogenic actions. However, the role of HGF in EPC mobilization has not been clearly described yet. We investigated the effect of HGF on mobilizing EPCs and on angiogenesis in elastase-induced lung injury. HGF significantly increased the triple-positive (Sca-1(+), Flk-1(+), and c-kit(+)) fraction in peripheral mononuclear cells in mice. The bone marrow-derived cells were recruited into the injured lungs, where they differentiated to capillary endothelial cells. HGF induced proliferation of both bone marrow-derived and resident endothelial cells in the alveolar wall. In conclusion, the present study suggests that HGF induces EPC mobilization from the bone marrow and enhances the proliferation of endothelial cells in vivo. These complex effects induced by HGF orchestrate pulmonary regeneration in emphysematous lung parenchyma.     

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.     

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.     

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.     

The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

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.