细胞因子对内皮祖细胞功能影响的研究进展

内皮祖细胞(EPCs)是一种具有较强增殖能力的前体细胞,血管损伤或者缺血会刺激骨髓EPCs动员,迁移、归巢于相应的靶位,然后分化为内皮细胞(ECs),从而参与血管修复和血管新生。因此,EPCs的成功发现为缺血性和血管损伤性疾病的治疗提供了新策略。但是EPCs存在动员率低、靶向性较差和功能不全等问题。大量研究显示细胞因子对EPCs的动员、归巢、增殖和分化等均起着重要的调节作用,同时,通过调控细胞因子能改善EPCs的功能活性,因此选择合适的细胞因子来提高EPCs功能变得非常重要。现总结了近年来细胞因子对EPCs功能影响的研究进展,并提出有待解决的问题和作一定的展望。     

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。     

血管再生中的内皮祖细胞

循环血液里存在一种被称为内皮祖细胞(endothelial progenitor cells,EPCs)的祖细胞亚群,具有在体内外分化为成熟内皮细胞的能力。根据内皮祖细胞与其他血液细胞的粘附能力的差异和内皮祖细胞的抗原特异性,内皮祖细胞可通过贴壁培养和免疫磁珠筛选而分离获得。内皮祖细胞可特异性表达三种祖细胞分子标志：CD133、CD34和血管内皮生长因子受体-2。当内皮祖细胞分化为成熟内皮细胞后,血小板内皮细胞粘附分子-1(CD31)、血管内皮粘附素(VE-cadherin,又称CD144)和Ⅷ因子(vWF)表达将上调。越来越多的证据显示,内皮祖细胞有利于体内内皮损伤后修复和血管再生。我们的研究发现,内皮祖细胞可修复apoE-缺陷小鼠血管移植物中的损伤内皮并且在动脉血管外膜中存在大量的血管祖细胞。然而,在机体的血管再生和动脉硬化的形成进程中,这些内皮祖细胞的作用和机制还不太明确。另外,有关机体内相应心血管疾病危险因素是如何影响内皮祖细胞功能的机制也不清楚。因此,对内皮祖细胞的归巢、释放和粘附机制的进一步深入研究将有助于人们探索内皮祖细胞的基础理论和临床应用价值。     

内皮祖细胞的生物学特性及其临床应用前景

内皮祖细胞（Endothelial Progenitor Cells,EPCs）是内皮细胞（endothelial cells,ECs）的前体细胞,即能分化为成熟ECs的祖细胞,它在血管内皮再生中发挥着重要作用。随着EPCs研究的深入,其在临床诊断、预后判断和各种缺血性疾病的治疗方面将会有广阔的应用前景。然而,关于EPCs的定义、来源、表面标记以及培养鉴定方法目前仍存在争议。     

内皮祖细胞(EPCs)研究进展

组织工程血管以及组织工程化组织的血管化因目前内皮种子细胞扩增能力和生物活力的不足而受到限制。EPCs(内皮祖细胞)是内皮细胞的前体细胞。在胚胎期,内皮细胞系与造血细胞系来源于血岛内共同的祖先细胞;出生后,EPCs存在于骨髓,并可被转移至外周血,参与缺血组织的血管重建和血管的内膜化。因此EPCs有望成为今后组织工程内皮种子细胞的重要来源。     

血管内皮祖细胞与炎性因子相关性的研究进展

内皮祖细胞(EPCs)是一种能直接分化为血管内皮细胞的前体细胞,不仅参与胚胎期的血管发生,还存在于骨髓、外周血和脐血中,在成体血管新生和受损内膜的再内皮化中发挥重要作用.然而,血管发生和炎症反应是两个密切联系的过程,血管损伤通常伴有局部促炎症介质的释放.多种炎症介质通过不同信号通路影响内皮祖细胞的数量和功能,从而影响损伤血管的修复和再生,参与炎症反应的发生和发展.本文结合近年来的研究进展,就EPCs与炎性相关因子如C-反应蛋白、肿瘤坏死因子-α、白细胞介素-1β、血管生成素、基质细胞衍生因子-1等的关系作一综述.     

血管内皮祖细胞与糖尿病微血管病变研究进展

糖尿病微血管病变严重影响了患者生活质量,是患者致死致残主要原因。微血管病变主要表现在视网膜、肾、神经、心肌组织。微血管病变的机制尚未完全清楚,近年越来越多研究发现血管内皮祖细胞(endothelial progenitor cells,EPCs)是该病发病重要原因。EPCs有分化为成熟的内皮细胞并且参与新血管形成和新生的能力。正常情况下内皮损失和EPCs对内皮的修复作用处于动态平衡状态,一旦EPCs受损,内皮损害和修复之间的平衡被打破,内皮层的完整性遭到破坏,必然参与糖尿病血管病变的发生发展。国内外大量研究证明糖尿病合并大血管病变EPCs数目功能改变,而糖尿病合并微血管病变EPCs的怎样变化?本文就EPCs与糖尿病微血管病变的关系进行系统综述。     

载脂蛋白A损伤小鼠骨髓源性EPCs血管发生能力及其机制

改善血流、促进血管新生是缺血性外周血管疾病的重要治疗措施．由于载脂蛋白A(ApoA)与纤溶酶原(plasminogen,Plg)具有75%～98%的结构同源性,因此,ApoA也可能通过类似Plg的方式抑制内皮祖细胞(endothelial progenitor cells,EPCs)增殖、黏附及迁移而影响血管发生的能力．本文研究ApoA对EPCs 血管发生的影响及机制．为了编码人ApoA全长cDNA序列的pSG-5表达载体,转染COS-7细胞株后进行培养,收集培养液,免疫亲和层析法分离纯化ApoA蛋白;从转ApoA基因小鼠、野生型对照鼠及正常对照鼠骨髓分离培养EPCs,经ApoA处理后移植下肢缺血实验小鼠,于移植后第3、7、14天后观察ApoA对EPCs黏附、迁移及血管发生能力的影响．研究发现,ApoA能显著降低 EPCs的黏附、迁移能力,Matrigel胶上,EPCs血管腔样结构严重破坏,体内实验揭示,EPCs归巢至ApoA转基因小鼠缺血组织血管周围的数量及毛细血管数量显著减少．结果表明,ApoA能损伤EPCs的黏附、迁移及归巢,最终损伤EPCs的血管发生能力．     

内皮祖细胞及其在组织工程中的应用

目前,组织工程化血管的构建和工程化组织器官的血管化因内皮种子细胞的扩增能力不足和生物活性不强而受到限制。内皮祖细胞(EPC)是内皮细胞的前体细胞。出生后,EPC主要存在于骨髓,可向外周血液缓慢释放,参与机体缺血组织的血管重建和损伤血管的重新内皮化。现对EPC的来源、分布、表型特征、动员、分化、归巢、分离、培养与鉴定等生物学特性和EPC在组织工程中的应用进行了全面的综述,并指出目前存在的问题和研究方向。     

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。     

Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance

Bone marrow and peripheral blood of adults contain a special sub-type of progenitor cells which are able to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization. These angiogenic cells have properties of embryonal angioblasts and were termed endothelial progenitor cells (EPCs). In general, three surface markers (CD133, CD34 and the vascular endothelial growth factor receptor-2) characterize the early functional angioblast, located predominantly in the bone marrow. Later, when migrating to the systemic circulation EPCs gradually lose their progenitor properties and start to express endothelial marker like VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. The number of circulating EPCs in healthy subjects is rather low and a variety of conditions or factors may further influence this number. In the context of possible therapeutic application of EPCs recent clinical studies employing these cells for neo-vascularization of ischemic organs have just been published. However, the specificity of the observed positive clinical effects, the mechanisms regulating the differentiation of EPCs and their homing to sites of injured tissue remain partially unknown at present.     

内皮祖细胞对急性肺损伤保护作用的研究进展

内皮祖细胞（EPC）是一种多潜能细胞,主要来源于骨髓。外周血EPC可以参与修复多种血管内皮细胞损伤的疾病。目前研究证实EPC通过动员、迁移、归巢和分化等步骤在受损的肺组织处参与内皮细胞修复,调节失控的炎症反应,增强抗氧化能力,对修复和维持肺泡毛细血管屏障的完整性起着重要作用。EPC在心血管疾病和组织工程领域应用研究的成功,为EPC在急性肺损伤的治疗提供了新的思路。     

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

Role of L-selectin in the vascular homing of peripheral blood-derived endothelial progenitor cells

Ex vivo expanded endothelial progenitor cells (EPCs) represent a new potential approach for the revascularization of ischemic sites. However, local accumulation of infused EPCs in these sites is poor, and the mechanisms responsible for their homing are largely unknown. We observed the expression of L-selectin, an adhesion receptor that regulates lymphocyte homing and leukocyte rolling and migration, on ex vivo expanded blood-derived human EPCs. When EPCs were subcloned in SV40-T large Ag-transfected isolates, the copresence of L-selectin and endothelial lineage markers was confirmed. We therefore demonstrated that the expression of L-selectin by EPCs was functional because it mediates interaction with a murine endothelial cell line (H.end) expressing L-selectin ligands by way of transfection with alpha(1,3/4)-fucosyltransferase. Indeed, adhesion of EPCs after incubation at 4 degrees C on a rotating platform was enhanced on alpha(1,3/4)-fucosyltransferase-transfected H.end cells compared with control vector-transfected cells, and treatment with anti-L-selectin Abs prevented this event. We then studied the role of L-selectin in EPC homing in vivo. H.end cells were implanted s.c. in SCID mice to form endothelioma tumors, and EPCs were subsequently i.v. injected. L-selectin+ EPCs localized into alpha(1,3/4)-fucosyltransferase-transfected endothelial tumors to a greater extent than in control tumors, and they were able to directly contribute to tumor vascularization by forming L-selectin+ EPC-containing vessels. In conclusion, our results showed that a mechanism typical of leukocyte adhesion is involved in the vascular homing of EPCs within sites of selectin ligand expression. This observation may provide knowledge about the substrate to design strategies to improve EPC localization in damaged tissues.     

mESC衍生内皮祖细胞定向分化为血管内皮细胞促伤口愈合

缺血性功能障碍是重要的全球健康问题。血管内皮细胞 (vascular endothelial cell, VEC) 在血管生成和创面修复中发挥关键作用,血管重建不足可导致慢性不愈合伤口。因此,了解有效的血管内皮细胞生成策略有助于受损组织中的血管再生。胚胎干细胞 (embryonic stem cell, ESC) 在组织的内皮化研究中应用广泛。内皮祖细胞 (endothelial progenitor cell, EPC) 是血管内皮细胞发育中不可或缺的部分。本研究目的在于找到一种小鼠胚胎干细胞 (mouse embryonic stem cell, mESC) 衍生为内皮祖细胞的快速、易筛选且高重复性的方法,并从内皮祖细胞定向分化中获得存活率高和功能性好的血管内皮细胞。结果表明,胚胎干细胞通过10 ng/mL VEGF和5 ng/mL bFGF定向诱导分化为增殖能力强的“铺路石”样祖细胞。同时,差异贴壁法有助于EPC的筛选。而EPC可诱导3 d的祖细胞高表达CD133和CD34(相对表达量分别为0.88 ± 0.04和2.12 ± 0.02);采用acctuse酶消化祖细胞,并在50 ng/mL VEGF和25 ng/mL bFGF的条件下诱导7 d分化为血管内皮样细胞,该细胞不仅高表达内皮细胞标志基因CD31、CD144、LAMA5、Tek、KDR和vWF,高表达标志蛋白CD31、CD144、LAMA5(相对表达量分别为1.07 ± 0.03、0.60 ± 0.02和0.70 ± 0.02),而且具有良好的迁移、成管和Weibel Palade (W-P) 小体形成能力。随后,将PBS、EPC和VEC分别应用于大小相同的创面治疗,EPC和VEC均能加快组织愈合程度(相对愈合率分别为78.93 ± 75.35%、95.57 ± 83.73%和100.00 ± 0.00%),VEC明显增强了伤口的血管生成能力和炎症反应。该研究初步证实,mESC衍生的EPC定向诱导7 d后可分化为血管内皮细胞。此内皮细胞具有较好的组织修复功能,干细胞促进血管生成的生理途径有望成为组织重塑的新靶点。     

Moderate dose insulin promotes function of endothelial progenitor cells

EPCs (endothelial progenitor cells) regenerate the vascular endothelial cells and keep the integrity of the vascular endothelium and thus may retard the onset of atherosclerosis. Steady state levels of EPCs in the circulation were found to be correlated with cardiovascular event risks. Given the close relationship between insulin and the cardiovascular system, we tested the long-term effects of moderate-dose insulin treatment on bone marrow-derived EPCs. Rat bone marrow EPCs were exposed to various levels of insulin under normal (5 mmol/l) or high (40 mmol/l) glucose conditions for 7 days. Insulin at levels near the physiological range (0.1, 1 nmol/l) up-regulated EPCs proliferation, stimulated NO (nitric oxide) production and reduced EPC senescence and ROS (reactive oxygen species) generation under both normal- and high-glucose conditions. Glucose exerted deleterious effects on EPCs contrary to insulin. Western blot analysis suggested concomitant decrease of Akt phosphorylation and eNOS (endothelial nitric oxide synthase) expression by high-glucose treatment and increase with insulin administration. Thus, insulin promoted several activities of EPCs, which suggested a potential endothelial protective role of insulin. Akt/eNOS pathway may be involved in the modulation of EPCs function by glucose and insulin.     

Directing migration of endothelial progenitor cells with applied DC electric fields

Naturally-occurring, endogenous electric fields (EFs) have been detected at skin wounds, damaged tissue sites and vasculature. Applied EFs guide migration of many types of cells, including endothelial cells to migrate directionally. Homing of endothelial progenitor cells (EPCs) to an injury site is important for repair of vasculature and also for angiogenesis. However, it has not been reported whether EPCs respond to applied EFs. Aiming to explore the possibility to use electric stimulation to regulate the progenitor cells and angiogenesis, we tested the effects of direct-current (DC) EFs on EPCs. We first used immunofluorescence to confirm the expression of endothelial progenitor markers in three lines of EPCs. We then cultured the progenitor cells in EFs. Using time-lapse video microscopy, we demonstrated that an applied DC EF directs migration of the EPCs toward the cathode. The progenitor cells also align and elongate in an EF. Inhibition of vascular endothelial growth factor (VEGF) receptor signaling completely abolished the EF-induced directional migration of the progenitor cells. We conclude that EFs are an effective signal that guides EPC migration through VEGF receptor signaling in vitro. Applied EFs may be used to control behaviors of EPCs in tissue engineering, in homing of EPCs to wounds and to an injury site in the vasculature.     

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells

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.