Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.     

Adult progenitor cells in vascular remodeling during atherosclerosis

The mobilization and recruitment of bone marrow-derived, circulating or tissue resident progenitor cells giving rise to smooth muscle-like cells have been implicated in neointima hyperplasia after arterial injury and in accelerated forms of arterial lesion formation, e.g., transplant arteriopathy or graft vasculopathy. By contrast, convincing evidence has emerged that the vascular homing of endothelial progenitor cells (EPCs) contributes to endothelial recovery, thus limiting neointima formation after arterial injury. In the chronic context of primary atherosclerosis, plaque progression and destabilization, a more complex picture has become apparent. In patients with coronary artery disease, the number and function of EPCs have been linked with an improved endothelial function or regeneration, but have been inversely correlated with cardiovascular risk. In animal models, however, the injection of bone marrow cells or EPCs, or the application of stem-cell mobilizing factors, have been associated with an exacerbation of atherosclerosis and unstable plaque phenotypes, whereas the contribution of bone marrow-derived smooth muscle progenitors to primary atherosclerosis appears to be rather confined. Here, we discuss crucial biochemical cues, namely chemokines, adhesion molecules, growth factors and pharmacological means that guide and control the context-specific mobilization, recruitment and fate of vascular progenitor cells in arterial remodeling during atherosclerosis.     

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。     

Ambivalence of progenitor cells in vascular repair and plaque stability

PURPOSE OF REVIEW: To discuss crucial cues (chemokines, adhesion molecules and pharmacological means) that guide and control the context-specific mobilization, recruitment and fate of circulating progenitor cells in arterial repair and plaque stability. RECENT FINDINGS: The mobilization and recruitment of bone marrow derived or resident progenitor cells giving rise to smooth muscle cells have been implicated in accelerated forms of primary plaque formation and neointimal hyperplasia after arterial injury. By contrast, convincing evidence has emerged that the arterial homing of endothelial progenitor cells contributes to endothelial recovery and thereby limits neointimal growth after endothelial denudation. In the chronic context of primary atherosclerosis, plaque progression and destabilization, a more complex picture has become apparent. Clinically, the number and function of endothelial progenitor cells have been linked with an improved endothelial function or regeneration and have been frequently inversely correlated with cardiovascular risk (factors). In animal models, however, the injection of bone marrow cells or endothelial progenitor cells, as well as the application of stem-cell mobilizing factors, have been associated with an exacerbation of atherosclerosis and unstable plaque phenotype, whereas the contribution of smooth muscle progenitors to primary atherosclerosis appears to be more confined to supporting plaque stability. SUMMARY: Considering the balance between distinct circulating vascular progenitor cells and identifying mechanisms for selective control of their mobilization and homing appears crucial to improve prediction and to directly modulate endogenous vascular remodeling processes.     

Endothelial progenitor cells in cardiovascular diseases

Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells(EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vas-culogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk fac-tors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardio-vascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evalu-ate the challenges facing EPC research and how these may be overcome.     

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.     

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。     

Angiogenic activity of endothelial progenitor cells through angiopoietin-1 and angiopoietin-2

Angiogenesis is a regulated process involving the proliferation, migration, and remodeling of different cell types particularly mature endothelial cells and recently discovered progenitor cells, named as endothelial progenitor cells (EPCs). Up to now, many attempts have been made to understand the dynamic balance of pro- and anti-angiogenic factors on EPCs on different milieu. It has been accepted that Ang-1, -2 and Tie-1, -2 signaling play a key role on angiogenesis pathways in endothelial lineage cells. In the current experiment, the angiogenic/angio-modulatory potency of Ang-1 and -2 was investigated on isolated EPCs. Freshly isolated EPCs were exposed to different concentrations of Ang-1 and -2 (25 and 50?ng/ml) over a course of 7 and 14 days. Corroborating to our results, a superior effect of Ang-1 on angiogenic properties, including an increased concentration of vascular endothelial growth factor, in vitro tubulogenesis, EPC migratory, Tie-2 expression and clonogenicity, was determined. A large amount of positive mature endothelium markers was achieved in EPCs being-exposed to Ang-1 peptide. Nonetheless, the number of CD133 positive cells increased in the presence of Ang-2. Collectively, we conclude that Ang-1 potentially induces functional and mature vascular-like behavior in EPCs more than Ang-2.     

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.     

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。     

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.     

Endothelial progenitor cells: identity defined?

In the past decade, researchers have gained important insights on the role of bone marrow (BM)-derived cells in adult neovascularization. A subset of BM-derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ , a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133⁺ cells or CD45⁺ haematopoietic cells.     

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(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。     

猪外周血内皮祖细胞的分离培养和鉴定

从猪外周血分离出单个核细胞,置于EGM-2培养基中培养,通过挑选细胞集落并对之进行免疫组织化学染色和荧光染色来鉴定内皮祖细胞。结果显示猪的内皮祖细胞为长梭形或纺锤形并呈集落生长,能够吞噬已酰化低密度脂蛋(ac-LDL)并结合凝结素BS-1,同时具有内皮细胞标志CD31、flk-1和von willebrand factor(vWF)。这些结果表明能够从猪的外周血中分离培养出内皮祖细胞,为自体内皮祖细胞移植促进猪慢性心肌缺血模型血管新生的研究打下了基础。     

Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood

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.     

Enhancing endothelial progenitor cell for clinical use

Circulating endothelial progenitor cells (EPCs) have been demonstrated to correlate negatively with vascular endothelial dysfunction and cardiovascular risk factors. However, translation of basic research into the clinical practice has been limited by the lack of unambiguous and consistent definitions of EPCs and reduced EPC cell number and function in subjects requiring them for clinical use. This article critically reviews the definition of EPCs based on commonly used protocols, their value as a biomarker of cardiovascular risk factor in subjects with cardiovascular disease, and strategies to enhance EPCs for treatment of ischemic diseases.     

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。     

Gene modification with integrin-linked kinase improves function of endothelial progenitor cells in pre-eclampsia in vitro

Integrin-linked kinase (ILK), a multifunctional serine-threonine protein kinase, has been shown to have implications for the treatment of ischemia vascular diseases by promoting angiogenesis in various tissues. However, whether this kinase has therapeutic potential in pre-eclampsia is not well studied. In this report, we determined the changes in the production and action of ILK on endothelial progenitor cells (EPCs) isolated from patients with pre-eclampsia. The effects of ILK transfection on proliferation, migration, and angiogenesis of EPCs were investigated. We showed that EPCs transfected with the ILK gene expressed high levels of ILK protein and mRNA. Transfection with ILK also enhanced the proliferative, migratory, and angiogenic capabilities of EPCs, and promoted the production of VEGF. These results suggest that ILK gene transfection is an effective approach to augment angiogenic properties of EPCs in vitro and providing basis for clinical cell-based gene therapy in patients with pre-eclampsia.