Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.     

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。     

Progenitor cells in vascular repair

PURPOSE OF REVIEW: A common characteristic of all types of vascular disease is endothelial dysfunction/damage followed by an inflammatory response. Although mature endothelial cells can proliferate and replace damaged cells in the vessel wall, recent findings indicate an impact of stem and progenitor cells in repair process. This review aims to briefly summarize the recent findings in stem/progenitor cell research relating to vascular diseases, focusing on the role of stem/progenitor cells in vascular repair. RECENT FINDINGS: It has been demonstrated that endothelial progenitor cells present in the blood have an ability to repair damaged arterial-wall endothelium. These cells may be derived from a variety of sources, including bone marrow, spleen, liver, fat tissues and the adventitia of the arterial wall. In response to cytokine released from damaged vessel wall and adhered platelets, circulating progenitor cells home in on the damaged areas. It was also reported that the adhered progenitor cells can engraft into endothelium and may differentiate into mature endothelial cells. SUMMARY: Vascular progenitor cells derived from different tissues have an ability to repair damaged vessel, in which the local microenvironment of the progenitors plays a crucial role in orchestrating cell homing and differentiation.     

Augmentation of Neovascularizaiton in Hindlimb Ischemia by Combined Transplantation of Human Embryonic Stem Cells-Derived Endothelial and Mural Cells

Background

We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration.

Methods and Results

After the expansion of human VPC-derived vascular cells, we transplanted these cells to nude mice with hindlimb ischemia. The blood flow recovery and capillary density in ischemic hindlimbs were significantly improved in human VPC-derived EC-transplanted mice, compared to human peripheral and umbilical cord blood-derived endothelial progenitor cells (pEPC and uEPC) transplanted mice. The combined transplantation of human VPC-derived EC and MC synergistically improved blood flow of ischemic hindlimbs remarkably, compared to the single cell transplantations. Transplanted VPC-derived vascular cells were effectively incorporated into host circulating vessels as EC and MC to maintain long-term vascular integrity.

Conclusions

Our findings suggest that the combined transplantation of human ES cells-derived EC and MC can be used as a new promising strategy for therapeutic vascular regeneration in patients with tissue ischemia.     

VEGF-R2 and neuropilin-1 are involved in VEGF-A-induced differentiation of human bone marrow progenitor cells

Tumor growth and metastasis require the generation of new blood vessels, a process known as neo-angiogenesis. Recent studies have indicated that early tumor vascularization is characterized by the differentiation and mobilization of human bone marrow cells. Vascular endothelial growth factor-A (VEGF-A) is one of the growth factors, which enhances their differentiation into endothelial cells, but little is known about the implication of the VEGF-receptor tyrosine kinases and about the implication of the VEGF-R co-receptor, neuropilin-1, in this process. In this context, the identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells was investigated in order to define the pharmaceutical targets involved in tissue vascularization associated with this process. For this purpose, an in vitro model of differentiation of human bone marrow AC133+ (BM-AC133+) cells into vascular precursors was used. In this work, we have demonstrated for the first time that the effect of VEGF-A on BM-AC133+ cells relies on an early action of VEGF-A on the expression of its tyrosine kinase receptors followed by an activation of a VEGF-R2/neuropilin-1-dependent signaling pathway. This signaling promotes the differentiation of BM-AC133+ cells into endothelial precursor cells, followed by the proliferation of these differentiated cells. Altogether, these results strongly suggest that VEGF inhibitors, acting at the level of VEGF-R2 and/or neuropilin-1, by inhibiting differentiation and proliferation of these cells, could be potentially active compounds to prevent progenitor cells to be involved in tumor angiogenesis leading to tumor growth.     

内皮祖细胞分离培养及应用技术新进展

内皮祖细胞(Endothelial progenitor cells,EPCs)是能够增殖、迁移、粘附并分化为血管内皮细胞潜能的一种原始细胞,在修复血管内皮和促进血管生成中具有重要的作用。EPC来源于骨髓,存在于骨髓、外周血、脐带血,新研究表明在脂肪组织、心肌中也能发现EPC的存在。EPC与干细胞的细胞表面标志物相似,功能上亦接近干细胞,但不具有自我更新的功能。近年来EPC已成为热点问题,对疾病诊断,预后判断和靶向治疗方面发挥重要作用,在冠状动脉粥样硬化性疾病、糖尿病血管病变、恶性肿瘤等治疗中全身或局部注射EPC具有更广泛的前景和应用价值。但关于分离培养EPC的方法及细胞表面标志物不完全相同,报道较少,至今尚没有形成统一的标准,本文就对于内皮祖细胞基本现状、分离培养技术、分选鉴定及临床应用方面做一综述。     

Endothelial progenitor cells in health and disease

There is currently great excitement and expectation in the stem cell community following the discovery that multipotent stem cells can be cultured from human fetal tissue and retain their ability to give rise to a variety of differentiated cell types found in all three embryonic germ layers. Although the earliest sites of hematopoietic cell and endothelial cell differentiation in the yolk sac blood islands were identified about 100 years ago, cells with hemangioblast properties have not yet been identified in vivo. Endothelial cells differentiate from angioblasts in the embryo and from endothelial progenitor cells, mesoangioblasts and multipotent adult progenitor cells in the adult bone marrow. Circulating endothelial progenitor cells (EPC) have been detected in the circulation after vascular injury and during tumor growth. The molecular and cellular mechanisms underlying EPC recruitment and differentiation are not yet understood, and remain as one of the central issues in stem cell biology. For many years, the prevailing dogma stated that the vessels in the embryo develop from endothelial progenitors, whereas sprouting of vessels in the adult results only from division of differentiated endothelial cells. Recent evidence, however, indicates that EPC contribute to vessel growth in the embryo and in ischemic, malignant or inflammed tissues in the adult, and can even be therapeutically used to stimulate vessel growth in ischemic tissues.     

Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration

Emerging evidence suggests that bone marrow-derived endothelial, hematopoietic stem and progenitor cells contribute to tissue vascularization during both embryonic and postnatal physiological processes. Recent preclinical and pioneering clinical studies have shown that introduction of bone marrow-derived endothelial and hematopoietic progenitors can restore tissue vascularization after ischemic events in limbs, retina and myocardium. Corecruitment of angiocompetent hematopoietic cells delivering specific angiogenic factors facilitates incorporation of endothelial progenitor cells (EPCs) into newly sprouting blood vessels. Identification of cellular mediators and tissue-specific chemokines, which facilitate selective recruitment of bone marrow-derived stem and progenitor cells to specific organs, will open up new avenues of research to accelerate organ vascularization and regeneration. In addition, identification of factors that promote differentiation of the progenitor cells will permit functional incorporation into neo-vessels of specific tissues while diminishing potential toxicity to other organs. In this review, we discuss the clinical potential of vascular progenitor and stem cells to restore long-lasting organ vascularization and function.     

TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However, the molecular mechanisms that regulate their differentiation, proliferation, and endothelial sheet formation remain to be elucidated. Here, we show that members of the transforming growth factor (TGF)-beta superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5-days postcoitum embryos. TGF-beta and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-beta and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover, SB-431542 up-regulated the expression of claudin-5, an endothelial specific component of tight junctions. These results suggest that endogenous TGF-beta/activin signals play important roles in regulating vascular growth and permeability.     

Differentiation of stem/progenitor cells into vascular cells in response to fluid mechanical forces

Vascular functions are regulated not only by chemical mediators, such as hormones, cytokines, and neurotransmitters, but by mechanical hemodynamic forces generated by blood flow and blood pressure. The mechanical force-mediated regulation is based on the ability of vascular cells, including endothelial cells and smooth muscle cells, to recognize fluid mechanical forces, i.e., the shear stress produced by flowing blood and the cyclic strain generated by blood pressure, and to transmit the signals into the cell interior, where they trigger cell responses that involve changes in cell morphology, cell function, and gene expression. Recent studies have revealed that immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells, as well as adult vascular cells, respond to fluid mechanical forces. Shear stress and cyclic strain promote the proliferation and differentiation of EPCs and ES cells into vascular cells and enhance their ability to form new vessels. Even more recently, attempts have been made to apply fluid mechanical forces to EPCs and ES cells cultured on polymer tubes and develop tissue-engineered blood vessel grafts that have a structure and function similar to that of blood vessels in vivo. This review summarizes the current state of knowledge concerning the mechanobiological responses of stem/progenitor cells and its potential applications to tissue engineering.     

Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels

Vasculogenesis, the in-situ assembly of angioblast or endothelial progenitor cells (EPCs), may persist into adult life, contributing to new blood vessel formation. However, EPCs are scattered throughout newly developed blood vessels and cannot be solely responsible for vascularization. Here, we identify an endothelial progenitor/stem-like population located at the inner surface of preexisting blood vessels using the Hoechst method in which stem cell populations are identified as side populations. This population is dormant in the steady state but possesses colony-forming ability, produces large numbers of endothelial cells (ECs) and when transplanted into ischaemic lesions, restores blood flow completely and reconstitutes de-novo long-term surviving blood vessels. Moreover, although surface markers of this population are very similar to conventional ECs, and they reside in the capillary endothelium sub-population, the gene expression profile is completely different. Our results suggest that this heterogeneity of stem-like ECs will lead to the identification of new targets for vascular regeneration therapy.     

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.     

Engineering blood vessels from stem cells: recent advances and applications

Endothelial cells organized into blood vessels are critical for the formation and maintenance of most tissues in the body and are involved in regulating physiological processes such as angiogenesis, inflammation and thrombosis. Endothelial cells are of great research interest, because of their potential to treat vascular diseases and to stimulate the growth of ischaemic tissue. They can be used to engineer artificial vessels, repair damaged vessels, and to induce the formation of vessel networks in engineered tissues. For such clinical applications, proliferating human endothelial progenitor cells can be isolated from adult tissues or embryonic stem cells. Recently, these cells were successfully used to engineer single vessels and to stimulate capillary networks, both in vitro and in vivo.     

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。     

肝细胞生长因子对骨髓内皮祖细胞的动员作用

目的: 分析肝细胞生长因子(HGF)能否动员骨髓内皮祖细胞,以及动员的内皮祖细胞能否参与创伤修复时的血管新生和内皮修复.方法: 将腺病毒HGF载体(adenovirus vector encoding HGF gene, Ad-HGF)经尾静脉注射到Balb/c小鼠体内,用ELISA方法检测血浆HGF水平的变化;用流式细胞术检测外周血CD34 细胞含量变化;对外周血单个核细胞进行分离、培养,并对生长的细胞克隆进行内皮细胞表面标志Tie-2、vW因子的免疫组化检测.建立雌性小鼠CCl4肝损伤模型,静脉移植HGF处理后雄性小鼠外周血单个核细胞到其体内,4 W后利用原位杂交技术检测新生肝组织中是否存在雄性细胞.结果: 注射Ad-HGF能明显提高小鼠血浆的HGF水平,并使外周血中以CD34、Tie-2和vW因子等为标志的内皮祖细胞的数量显著增多.这些细胞参与肝损伤修复时的血管新生.结论: HGF对骨髓内皮祖细胞具有明显的动员作用.     

Adult 'endothelial progenitor cells'. Renewing vasculature

During embryogenesis, endothelial progenitor cells participate in the initial processes of primitive blood vessel formation (vasculogenesis). It has become evident that progenitors to vascular endothelial cells also exist in the adult. Endothelial progenitors normally reside in the adult bone marrow but may become mobilized into circulation by cytokine or angiogenic growth factor signals from the periphery, enter extravascular tissue, and promote de novo vessel formation by virtue of physically integrating into vessels and/or supplying growth factors (adult vasculogenesis). For that reason, autologous endothelial progenitors, mobilized in situ or transplanted, has become a major target of therapeutic revascularization approaches to ischemic disease and endothelial injury. Moreover, endothelial progenitors represent a potential target of strategies to block tumor growth.     

Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization

Adult bone marrow is a rich reservoir of hematopoietic and vascular stem and progenitor cells. Mobilization and recruitment of these cells are essential for tissue revascularization. Physiological stress, secondary to tissue injury or tumor growth, results in the release of angiogenic factors, including vascular endothelial growth factor (VEGF), which promotes mobilization of stem cells to the circulation, contributing to the formation of functional vasculature. VEGF interacts with its receptors, VEGFR2 and VEGFR1, expressed on endothelial and hematopoietic stem cells, and thereby promotes recruitment of these cells to neo-angiogenic sites, accelerating the revascularization process. The mobilization of stem cells from marrow is a dynamic process, regulated by shear stress imparted by blood flow, and the activation of metalloproteinases that induce the release of 'Kit ligand', facilitating egress from the marrow to the circulation. Identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells will open up new avenues for the design of clinical trials to accelerate tissue vascularization and organogenesis.     

HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell-derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1-PI3K-Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor-induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell-derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1-PI3K-Akt-HDAC3-p53-p21 pathway is crucial in such a process.     

Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell-derived VEGFR2(+) mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)-BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A-induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras(-/-) mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2(+) progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2(+) progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6-9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras-Erk signaling to direct endothelial specification of VEGFR2(+) vascular progenitor cells.