LFA-1 and VLA-4 involved in human high proliferative potential-endothelial progenitor cells homing to ischemic tissue

Cumulative evidences have revealed that endothelial progenitor cell (EPC) transplantation can promote the neovascularization in ischemic tissue, but the mechanism of EPCs homing to the site of ischemia is poorly understood. In this study, to investigate the mechanism of human umbilical cord blood-derived high proliferative potential-endothelial progenitor cells (HPP-EPCs) homing to ischemic tissue we evaluated the expression of lymphocyte function-associated antigen-1 (LFA-1, or CD11a/CD18) and very late antigen-4 (VLA-4, or CD49d/CD29) in EPCs and the changes of expression level of their ligands, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in ischemic tissue and performed the adhesion and migration assays to analyze the interaction between the receptors and ligands. Furthermore, we studied the roles of LFA-1 and VLA-4 in EPC homing in an ischemic model of mice. The results show that LFA-1 andVLA-4 were expressed in HPPEPCs and ICAM-1 and VCAM-1 were expressed in vessel endothelium in ischemic tissues. The pre-incubation of HPP-EPCs with neutralizing antibodies against CD11a or CD49d reduced adhesion and migration of HPP-EPCs in vitro and reduced recovery of hind-limb blood flow, capillary density and incorporation of HPP-EPC into ischemic tissues in vivo. Furthermore, the pre-incubation of HPP-EPCs with the combination of CD11a and CD49d antibodies led to synergistically negative effects on adhesion and transmigration of HPP-EPCs in vitro, and on the homing of HPP-EPCs to ischemic tissue and on neovascularization capacity in vivo. These results indicate that LFA-1 andVLA-4 are involved in HPP-EPC homing to ischemic tissues.     

Increased levels of circulating endothelial progenitor cells and circulating endothelial nitric oxide synthase in patients with gliomas

OBJECTIVE: Gliomas are among the highest vascularized tumors. We hypothesized that patients with gliomas have increased levels of circulating endothelial progenitor cells (EPCs) and circulating endothelial nitric oxide synthase (eNOS). METHODS: The fraction of EPCs was quantified by fluorescence-activated cell sorter analysis using anti-CD34, -CD133 and -KDR (kinase insert domain receptor) monoclonal antibodies in unselected peripheral blood samples of 32 patients with gliomas. Control groups included 47 patients with other central nervous system tumors or diseases, 10 patients with recent ischemic strokes, and 19 healthy blood donors. The circulating eNOS concentration of plasma was measured by a colorimetric assay in the same samples. In addition, CD34(+)CD105(+) KDR(+) and CD34(+)CD146(+)KDR(-) cell fractions were measured. RESULTS: The percentage of CD34(+)CD133(+)KDR(+) EPCs in the blood of glioma patients is significantly greater than that in the blood of patients with other central nervous system tumors or diseases (p = 0.003), stroke patients (p = 0.005), or healthy donors (p = 0.013). The plasma eNOS concentration is also significantly greater in glioma patients compared with each of the control groups (p < 0.001 for all groupwise comparisons). No significant differences in the levels of the EPCs or eNOS between any of the control groups were demonstrated. In the glioma patients, the level of eNOS correlated with the fraction of CD34(+)CD105(+)KDR(+) cells (r = 0.748; p = 0.008). INTERPRETATION: The data are suggestive of increased mobilization of EPCs contributing to neoplastic vasculogenesis in glioma. The increased levels of EPCs and eNOS in the peripheral blood of glioma patients trigger further investigations as to their value as independent parameters for use in clinical practice.     

Potential role of activated platelets in homing of human endothelial progenitor cells to subendothelial matrix

Endothelial progenitor cells (EPCs) mobilize from the bone marrow in response to tissue injury and participate in vascular repair. However, there is limited data about the homing mechanisms of EPCs to vascular injury sites. Recently animal experiments indicated that platelets play a role in recruitment of EPCs to injury sites. However, data on the possible interaction between platelets and EPCs within the human system are limited. We, therefore, examined in-vitro human platelet-EPC interaction under static and flow conditions. Human EPCs were isolated from donated buffy coats by magnetic microbeads and flow cytometry cell sorting using CD133 and VEGFR-2, respectively, as markers. Platelets were tested in the form of washed platelets, platelet rich plasma or whole blood. EPCs formed heterotypic aggregates with resting platelets under static conditions, an interaction that was greatly enhanced when platelets were activated by collagen, ADP or thrombin-activation peptide. The platelet-EPC interaction was inhibited by antibodies to P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1), but not by antibodies to glycoproteins Ib-IX-V or IIb/IIIa. When perfused over activated platelets under shear stress of 2.5 dyn/cm(2), EPCs tethered to platelayers and either adhered immediately or rolled a short distance before adhering. In addition, platelets promoted the colonization of adherent EPCs in culture conditions. Consistent with recent animal studies, these findings demonstrate that human EPCs interact in vitro with activated platelets under static and flow conditions, mediated through P-selectin-PSGL-1 interaction. This interaction may be a central mechanism for homing of EPCs to vascular injury sites.     

Expression of transcription factor Oct-4 and other embryonic genes in CD133 positive cells from human umbilical cord blood

A significant number of hematopoietic stem/progenitor cells (HSPC) in human umbilical cord blood could serve as a reservoir for the placental vasculature, yet, their morphological and functional features are not completely understood. Here, we describe the characterization of purified CD133(+) progenitor cells from umbilical cord blood, a subset of CD34(+) hematopoietic progenitors that were grown in proliferation medium containing Flt3-ligand, thrombopoietin and stem cell factor. Following isolation and enrichment of the CD133(+) cells by immunomagnetic cell sorting, they remained non-adherent for up to 40 days in culture and expressed different pluripotency markers including Sox-1, Sox-2, FGF-4, Rex-1 and Oct-4.Oct-4 expression was confirmed by laser-assisted single cell picking with subsequent quantitative real-time RT-PCR. The expression of Oct-4 indicates a pluripotent phenotype of CD133(+) cells and appears to be of functional relevance: After three weeks in endothelial differentiation medium, suspended cells became adherent, developed an endothelial cell-like morphology, bound fluoresceine isothiocyanate-labeled Ulex europaeus agglutinin-1, took up acetylated Di-LDL, and expressed other endothelial markers such as PECAM-1 or VEGFR-2. Concomitantly, Oct-4 expression was significantly reduced. Moreover, following treatment with retinoic acid, CD133(+) cells exhibited neural morphology associated with the expression of beta-III-tubulin. CD133(+) cells were found to express the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, detected by RT-PCR and immunocytochemistry. The recombinant human chorionic gonadotropin induced proliferation of the CD133(+) cells in a dose-specific manner. Our results indicate that CD133(+) HSPC from umbilical cord blood may have a greater differentiation potential than previously recognized and give rise to proliferative endothelial cells participating in placental vasculogenesis.     

Decreased level and defective function of circulating endothelial progenitor cells in children with moyamoya disease

Circulating endothelial progenitor cells (EPCs) play an important role in physiological and pathological neovascularization and may be involved in attenuating ischemic diseases. This study aimed to characterize circulating EPCs in moyamoya disease (MMD), one of the most common pediatric cerebrovascular diseases. Twenty‐eight children with MMD prior to any surgical treatment and 12 healthy volunteers were recruited. Peripheral blood mononuclear cells (PBMNCs) were isolated and cultured in endothelial cell growth medium. Temporal change of phenotype of cells was analyzed on days 0 and 7. The formation of EPC clusters was evaluated on day 7. The CD34⁺, CD133⁺, and KDR⁺ cells, and the number of EPC clusters was significantly reduced in children with MMD. In controls, CD34⁺ cells were significantly decreased on day 7 compared with day 0, but in MMD they were only slightly decreased. The change in KDR⁺ cells on day 7 compared with day 0 was the reverse of that for CD34⁺ cells. Functional assay of EPC demonstrated less tube formation and increased senescent‐like phenotype in children with MMD. Analysis of the circulating EPCs of MMD children reveals decreased level and defective function. This study suggests that circulating EPCs may be associated with MMD pathogenesis. © 2009 Wiley‐Liss, Inc.     

未破裂颅内动脉瘤患者外周血内皮祖细胞数量的研究

目的 研究颅内动脉瘤与外周血中内皮祖细胞(EPCs)数量的关系.方法 选择未破裂颅内动脉瘤患者24例和正常对照者16例,用密度梯度离心法获取外周血单个核细胞,加入培养基进行选择性培养,分析14 d后细胞形成的集落数,并对贴壁细胞进行细胞鉴定、分析和计数.激光共聚焦显微镜下观察其吞噬荆豆凝集素和乙酰化低密度脂蛋白的内皮功能;用流式细胞仪检测CD34、CD133和血管内皮生长因子受体2(VEGFR-2)三抗阳性细胞并计数.结果 颅内动脉瘤患者外周血EPCs计数较对照组明显减少,流式分析结果显示三抗阳性细胞数比例有显著性差异.结论 颅内动脉瘤患者外周血EPCs的数量较无动脉瘤者明显降低.     

Role of c-kit/Kit ligand signaling in regulating vasculogenesis

Mobilization into peripheral blood of bone marrow-derived cells including hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), is regulated by chemokines/cytokines. These cells can contribute to the formation of new blood vessels (vasculogenesis) under pathological conditions including atherosclerosis, wound healing and tumor growth. We will review how these cells are mobilized into circulation, and supplied to the sites, where vessel formation is needed (i.e. ischemic tissue or tumor bed). We will give evidence that matrix metallo-proteinase-9 mediated Kit ligand (Stem cell factor) processing is essential for cell mobilization induced by chemo-/cytokines, like vascular endothelial growth factor (VEGF), Placental growth factor (PlGF), stromal cell derived factor-1 (SDF-1). These studies may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting kit ligand mediated mobilization and homing of bone marrow-derived progenitor cells for cell therapy and cancer therapy.     

Increase in circulating CD34-positive cells in patients with angiographic evidence of moyamoya-like vessels.

Increasing evidence points to a role for circulating endothelial progenitor cells, including populations of CD34-positive (CD34(+)) cells, in maintenance of cerebral blood flow. In this study, we investigated the link between the level of circulating CD34(+) cells and neovascularization at ischemic brain. Compared with control subjects, a remarkable increase of circulating CD34(+) cells was observed in patients with angiographic moyamoya vessels, although no significant change was observed in patients with major cerebral artery occlusion (or severe stenosis) but without moyamoya vessels. Our results suggest that the increased level of CD34(+) cells associated with ischemic stress is correlated with neovascularization at human ischemic brain.     

脂联素转染的内皮祖细胞对糖尿病大鼠脑缺血再灌注损伤的保护作用

目的 探讨脂联素(adiponectin, APN)基因转染的内皮祖细胞(endothelial progenitor cells, EPCs)移植对2型糖尿病大鼠(type 2 diabetes mellitus, T2DM)脑缺血再灌注损伤(ischemic-reperfusion injury,I/R)的保护作用及其可能的作用机制。方法 4周龄SD雄性大鼠骨髓分离培养EPCs,随机分组并干预:未干预组(EPCs组)、转染APN基因的EPCs(LV-APN-EPCs组)和空白病毒EPCs(LV-EPCs组); 5周龄SD雄性大鼠70只,随机分为假手术组(sham组)、对照组(PBS组)、LV-APN-EPCs治疗组、LV-EPCs治疗组和EPCs治疗组,高脂结合链脲佐菌素诱导T2DM模型,线栓法造I/R模型,I/R损伤后1 h分别静脉注射1 mL PBS、PBS、 LV-APN-EPCs、LV-EPCs和EPCs,I/R前和I/R后的第1、7、14 d,各组进行mNss评分; 细胞治疗后第14 d,各组行脑组织HE染色、CD31+免疫荧光染色,ELISA法检测炎症因子IL-β和TNF-α的水平。结果 I/R后第7、14 d LV-APN-EPCs组的mNss评分明显小于LV-EPCs组、EPCs组和PBS组(P<0.05); 细胞治疗后第14 d,LV-APN-EPCs组神经元破坏较LV-EPCs组、EPCs组和PBS组明显减轻; CD31+免疫荧光染色显示LV-APN-EPCs治疗显著增加了I/R损伤区皮质的血管密度(P<0.05); ELISA显示LV-APN-EPCs组脑组织的炎症因子IL-β和TNF-α的水平明显较LV-EPCs组、EPCs组和PBS组低(P<0.05)。结论 LV-APN-EPCs治疗对T2DM大鼠脑I/R损伤发挥保护作用,其可能的作用机制是促进脑I/R损伤区血管再生和抑制炎症因子的表达。     

巴曲酶可影响人外周血内皮祖细胞定向分化及增殖能力

背景：近年来内皮祖细胞促进机体血管新生方面的重要作用已形成共识,但内皮祖细胞的定向分化和扩增的问题是影响其疗效和广泛开展的主要原因。 目的：观察人外周血内皮祖细胞体外分离、纯化、扩增和诱导分化为内皮细胞的可行性。 方法：用密度梯度离心法从外周血获取单个核细胞,接种于包被有人纤维连接蛋白的细胞培养板中,培养6 d后,收集贴壁细胞。以血管内皮细胞生长因子和碱性成纤维细胞生长因子对贴壁细胞进行诱导培养。另外,分别以0.05,0.1,0.2 BU/mL巴曲酶培养贴壁细胞,观察内皮祖细胞的增殖能力。 结果与结论：经血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导后的细胞形态上表现内皮细胞特征,经流式细胞仪测定表达血管内皮细胞特有表面标志CD31和vWF,说明诱导后的细胞为内皮细胞。巴曲酶在体外培养条件下可增加血内皮祖细胞的数量,以0.1 BU/mL浓度作用显著,且作用时间与血内皮祖细胞增殖能力的改善呈正相关。     

关于针刺治疗脑缺血疾病机制中血管新生途径的新思路

在针刺治疗脑缺血疾病临床疗效机制的研究中,内皮祖细胞等存在于成体内的干细胞参与机体损伤修复的作用已成为当前组织工程研究的热点之一。资料显示,针刺可以从多种途径缓解和改善脑缺血带来的损伤且具有明显的治疗效果,而血管新生可能是其中的重要途径之一。缺血性损伤会导致内皮祖细胞参与血管新生修复,多种细胞因子在这一过程中发挥作用,它们可动员并诱导内皮祖细胞归巢。现有研究表明,针刺可以影响其中部分因子的表达,这种内在联系为针刺治疗脑缺血机制的认识开辟了新的思路。     

缺血性脑血管病患者颈动脉粥样硬化程度与循环内皮祖细胞的相关性分析

目的探讨缺血性脑血管病患者颈动脉粥样硬化程度与循环内皮祖细胞(EPCs)的相关性。方法根据CD133和KDR标记,采用流式细胞仪检测缺血性脑血管病患者(包括TIA、急性脑梗死、颈动脉粥样硬化)和健康体检者外周血内皮祖细胞(EPCs)数量变化。结果缺血性脑血管病患者EPCs数量较对照组明显减少(P<0.01);随着颈动脉粥样硬化程度加重,EPCs数量呈降低趋势,颈动脉重度狭窄与轻度和中度狭窄相比有差异(P<0.05);急性脑梗死和TIA患者较单纯颈动脉粥样硬化患者EPCs数量明显增加(P<0.01)。结论缺血性脑血管病患者EPCs与颈动脉粥样硬化程度呈负相关,可反映颈动脉粥样硬化的程度;急性缺血可能会增加EPCs的动员。     

Difficulties in work-related activities among migraineurs are scarcely collected: results from a literature review

Circulating endothelial progenitor cells (EPCs) play a critical role in maintaining endothelial integrity and keeping vascular homeostasis. Previously, we reported that EPCs were involved in repair and remodeling of aneurismal wall. In the present study, we verified this hypothesis by investigating the proliferative ability and count of EPCs in peripheral blood of patients with unruptured intracranial aneurysms (UIAs). Twenty-four patients with UIAs (UIA group) and 24 negative controls (control group) were included in this study. Peripheral blood monocytes (PBMCs) were harvested and selectively cultured. The colony-forming ability of cultured cells was analyzed and the biological functions were examined by testing the adsorption of ulex europaeus agglutinin-1 labeled by fluorescein isothiocyanate and acetylated low-density lipoprotein internalization. The migratory and adhesive ability of cultured EPCs were assessed. In vitro cultured PBMCs were identified as EPCs by examining surface markers CD34, CD133 and vascular endothelial growth factor receptor 2 using flow cytometry. EPCs from UIA group possessed significantly decreased proliferative, migratory and adhesive capacities compared with EPCs from control group. Furthermore, EPCs count in UIA group was significantly decreased. Collectively, these results indicated that the circulating EPCs of UIA patients may be involved in intracranial aneurysm repair and remodeling.     

Intracranial transplantation of monocyte-derived multipotential cells enhances recovery after ischemic stroke in rats

Cell transplantation has emerged as a potential therapy to reduce the neurological deficits caused by ischemic stroke. We previously reported a primitive cell population, monocyte-derived multipotential cells (MOMCs), which can differentiate into mesenchymal, neuronal, and endothelial lineages. In this study, MOMCs and macrophages were prepared from rat peripheral blood and transplanted intracranially into the ischemic core of syngeneic rats that had undergone a left middle cerebral artery occlusion procedure. Neurological deficits, as evaluated by the corner test, were less severe in the MOMC-transplanted rats than in macrophage-transplanted or mock-treated rats. Histological evaluations revealed that the number of microvessels that had formed in the ischemic boundary area by 4 weeks after transplantation was significantly greater in the MOMC-transplanted rats than in the control groups. The blood vessel formation was preceded by the appearance of round CD31(+) cells, which we confirmed were derived from the transplanted MOMCs. Small numbers of bloodvessels incorporating MOMC-derived endothelial cells expressing a mature endothelial marker RECA-1 were detected at 4 weeks after transplantation. In addition, MOMCs expressed a series of angiogenic factors, including vascular endothelial growth factor, angiopoetin-1, and placenta growth factor (PlGF). These findings provide evidence that the intracranial delivery of MOMCs enhances functional recovery by promoting neovascularization in a rat model for ischemic stroke.     

Presence of endothelial progenitor cells, distinct from mature endothelial cells, within human CD146+ blood cells

CD146 is an adhesion molecule present on endothelial cells throughout the vascular tree. CD146 is also expressed by circulating endothelial cells (CECs) widely considered to be mature endothelial cells detached from injured vessels. The discovery of circulating endothelial progenitor cells (EPCs) originating from bone marrow prompted us to investigate whether CD146 circulating cells could also contains EPCs. We tested this hypothesis using an approach combining elimination of CECs by an adhesion step, followed by immunomagnetic sorting of remaining CD146+ cells from the non adherent fraction of cord blood mononuclear cells. When cultured under endothelial-promoting conditions, these cells differentiated as late outgrowth endothelial colonies: they grew as a cobblestone monolayer, were uniformly positive for endothelial markers and did not express leukocyte antigens. They highly proliferated and were expanded in long-term culture without alterations of their phenotypic and functional properties (Dil-ac-LDL uptake, wound repair, capillary-like network formation, and TNFalpha response). Moreover, these cells colonized a Matrigel plug in immunodeficient mice (NOD/SCID). Finally, using 4-color flow cytometry analysis of purified CD34+ cells, we clearly discriminated, CD146+ EPCs (CD146+ CD34+ CD45+ CD133+ or CD117+), and CD146+ CECs (CD146+ CD34+, CD45- CD133- or CD117-), both in cord and adult peripheral blood.The relative proportions of the two CD146+ subsets varied in patients with myocardial infarction as compared to healthy subjects. Our study establishes that, beside CECs, CD146+ circulating cells contain a subpopulation of EPCs with potential use in proangiogenic therapy. In addition, the dual measurement of CD146+ CECs and CD146+ EPCs offers a promising tool for monitoring vascular injury/regeneration processes in clinical situations.     

Circulating endothelial progenitor cells and depression: a possible novel link between heart and soul

Although depression is known to be an independent risk factor for cardiovascular disorders, the mechanisms behind this connection are not well understood. However, the reduction in the number of endothelial progenitor cells (EPCs) in patients with cardiovascular risk factors has led us to hypothesize that depression influences the number of EPCs. EPCs labeled with CD34, CD133 and vascular endothelial growth factor receptor-2 (VEGFR2) antibodies were counted by flow cytometry in the peripheral blood (PB) of 33 patients with a current episode of major depression and of 16 control subjects. Mature (CD34+/VEGFR2+) and immature (CD133+/VEGFR2+) EPC counts were decreased in patients (vs controls; P<0.01 for both comparisons), and there was a significant inverse relationship between EPC levels and the severity of depressive symptoms (P<0.01 for both EPC phenotypes). Additionally, we assayed the plasma levels of VEGF, C-reactive protein (CRP) and tumor necrosis factor (TNF)-alpha and observed significantly elevated TNF-alpha concentrations in patients (vs controls; P<0.05) and, moreover, a significant inverse correlation between TNF-alpha and EPC levels (P<0.05). Moreover, by means of a quantitative RT-PCR approach, we measured CD34, CD133 and VEGFR2 mRNA levels of PB samples and found a net trend toward a decrease in all the investigated EPC-specific mRNA levels in patients as compared with controls. However, statistical significance was reached only for VEGFR2 and CD133 levels (P<0.01 for both markers). This is the first paper that demonstrates evidence of decreased numbers of circulating EPCs in patients with a current episode of major depression.     

Contribution of bone marrow‐derived endothelial progenitor cells to neovascularization and astrogliosis following spinal cord injury

Spinal cord injury causes initial mechanical damage, followed by ischemia‐induced, secondary degeneration, worsening the tissue damage. Although endothelial progenitor cells (EPCs) have been reported to play an important role for pathophysiological neovascularization in various ischemic tissues, the EPC kinetics following spinal cord injury have never been elucidated. In this study, we therefore assessed the in vivo kinetics of bone marrow‐derived EPCs by EPC colony‐forming assay and bone marrow transplantation from Tie2/lacZ transgenic mice into wild‐type mice with spinal cord injury. The number of circulating mononuclear cells and EPC colonies formed by the mononuclear cells peaked at day 3 postspinal cord injury. Bone marrow transplantation study revealed that bone marrow‐derived EPCs recruited into the injured spinal cord markedly increased at day 7, when neovascularization and astrogliosis drastically occurred in parallel with axon growth in the damaged tissue. To elucidate further the contribution of EPCs to recovery after spinal cord injury, exogenous EPCs were systemically infused immediately after the injury. The administered EPCs were incorporated into the injured spinal cord and accelerated neovascularization and astrogliosis. These findings suggest that bone marrow‐derived EPCs may contribute to the tissue repair by augmenting neovascularization and astrogliosis following spinal cord injury. © 2012 Wiley Periodicals, Inc.     

绵羊骨髓来源内皮祖细胞的培养与鉴定*☆

背景：当今,组织工程化静脉瓣的研究刚刚起步,种子细胞的选择是其关键,内皮祖细胞可以作为组织工程化静脉瓣体外构建理想的种子细胞。 目的：通过体外培养与鉴定绵羊骨髓来源内皮祖细胞,探讨绵羊骨髓来源内皮祖细胞培养方法,为绵羊组织工程静脉瓣种子细胞选择提供实验基础。 方法：绵羊骨髓经条件培养基进行选择培养获取骨髓单个核细胞,传代扩增后用磁珠分选出CD133+细胞再培养,流式细胞检测确认分前细胞CD133的表达情况;分选传代后绘制细胞生长曲线观察细胞生长能力,用免疫细胞化学检测细胞特异性分子CD133,D34,VWF的表达情况;FITC标记BS-1-lectin和DiI标记ac-LDL标记细胞检测细胞吞噬功能。 结果与结论：绵羊骨髓单个核细胞原代培养2 d细胞开始贴壁,7 d细胞完全融合,传代后第2天进入对数生长期,传代3~5 d形成为典型的铺路石样,传代后第7 d细胞进入增殖平台期;细胞传代后磁珠分选率为12.6%,流式细胞仪检测显示CD133阳性率为12.64%;免疫细胞化学检测显示细胞呈CD133、CD34、血管性血友病因子阳性表达。细胞能同时吞噬FITC-labeled BS-1-lectin,DiI-ac-LDL阳性率达85.3%。证实实验成功地从绵羊骨髓单个核细胞中分离培养出内皮祖细胞。     

Regulation of endothelial progenitor cell homing after arterial injury

Adult bone marrow and peripheral blood contain sub-populations of vascular precursor cells, which can differentiate into mature endothelial cells and have therefore been commonly termed endothelial progenitor cells (EPCs). Although EPCs encompass rather heterogeneous cell sub-populations of multiple origins and localization, these cells were basically characterized by expression of progenitor markers and by the development of colony-forming units and late endothelial outgrowth with terminal differentiation into mature endothelial cells. Notably, functional studies in vivo have implied the contribution of EPCs to therapeutic reendothelialization and inhibition of neointimal growth following endothelial injury. In the context of this regenerative arterial remodeling, an adequate homing of EPCs plays a central role. This multi-step process of EPC mobilization, recruitment and firm adhesion is regulated by key angiogenic chemokines (CCL2, CXCL1, CXCL7, CXCL12) and their respective receptors (CCR2, CXCR2, CXCR4). Furthermore, the recruitment of circulating EPCs to sites of arterial injury is synchronized by activated platelets and adhesion molecules of the selectin and integrin family. Thus, translating this molecular knowledge to interventional cardiovascular medicine, such a detailed understanding in the complex regulation of EPC homing may be helpful for more effectively preventing "in-stent" stenosis by facilitating stent endothelialization.     

Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage

Breakdown of blood-brain barrier,formed mainly by brain microvascular endothelial cells(BMECs),represents the major cause of mortality during early phases of ischemic strokes.Hence,discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine.Although endothelial progenitor cells(EPCs)represent one such agents,their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells(OECs)as a result.Functional analyses of these cells,in the present study,demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel,prominent endothelial cell and angiogenesis markers,respectively.Further analyses by flow cytometry demonstrated that OECs expressed specific markers for sternness(CD34),immaturity(CD133)and endothelial cells(CD31)but not for hematopoietic cells(CD45).Like BMECs,OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes,suggesting their capacity to form tight junctions.Ischemic injury mimicked by concurrent deprivation of oxygen and glucose(4 hours)or deprivation of oxygen and glucose followed by reperfusion(20 hours)affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux,respectively.Wound scratch assays comparing the vasculoreparative capacity of cells revealed that,compared to BMECs,OECs possessed a greater proliferative and directional migratory capacity.In a triple culture model of BBB established with astrocytes,pericytes and BMEC,exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions.Taken together,these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain(neuro)vascular homeostasis during or after a cerebral ischemic injury.