Role of beta2-integrins for homing and neovascularization capacity of endothelial progenitor cells

The mechanisms of homing of endothelial progenitor cells (EPCs) to sites of ischemia are unclear. Here, we demonstrate that ex vivo-expanded EPCs as well as murine hematopoietic Sca-1+/Lin- progenitor cells express beta2-integrins, which mediate the adhesion of EPCs to endothelial cell monolayers and their chemokine-induced transendothelial migration in vitro. In a murine model of hind limb ischemia, Sca-1+/Lin- hematopoietic progenitor cells from beta2-integrin-deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the beta2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo. These results provide evidence for a novel function of beta2-integrins in postnatal vasculogenesis.     

基质细胞衍生因子1α预处理内皮祖细胞移植治疗糖尿病下肢缺血

背景:基质细胞衍生因子1α被证明可以促进内皮祖细胞归巢。目的:观察基质细胞衍生因子1α预处理内皮祖细胞移植治疗糖尿病模型鼠下肢缺血的疗效。方法:取雄性Wistar鼠30只,25只成功建立糖尿病模型大鼠,随机数字表法分为3组,对照组注入等量生理盐水,共培养组注射与基质细胞衍生因子1α共培养的内皮祖细胞,内皮祖细胞组注入未进行共培养的内皮祖细胞。结果与结论:MTT检测结果显示,基质细胞衍生因子1α能显著增加内皮祖细胞的增殖能力(P<0.01)。移植后第28天动脉造影显示共培养组缺血侧下肢动脉显影血管数多于内皮祖细胞组和对照组(P<0.05)。提示采用基质细胞衍生因子1α预处理内皮祖细胞,有利于使糖尿病大鼠缺血下肢血供改善,主要来自于新生血管形成和/或原有细小血管的代偿性增粗。     

基质细胞衍生因子1α预处理内皮祖细胞移植治疗糖尿病下肢缺血

背景：基质细胞衍生因子1α被证明可以促进内皮祖细胞归巢。目的：观察基质细胞衍生因子1α预处理内皮祖细胞移植治疗糖尿病模型鼠下肢缺血的疗效。方法：取雄性Wistar鼠30只,25只成功建立糖尿病模型大鼠,随机数字表法分为3组,对照组注入等量生理盐水,共培养组注射与基质细胞衍生因子1α共培养的内皮祖细胞,内皮祖细胞组注入未进行共培养的内皮祖细胞。结果与结论：MTT检测结果显示,基质细胞衍生因子1α能显著增加内皮祖细胞的增殖能力（P〈0.01）。移植后第28天动脉造影显示共培养组缺血侧下肢动脉显影血管数多于内皮祖细胞组和对照组（P〈0.05）。提示采用基质细胞衍生因子1α预处理内皮祖细胞,有利于使糖尿病大鼠缺血下肢血供改善,主要来自于新生血管形成和/或原有细小血管的代偿性增粗。     

外周血内皮祖细胞移植对肢体缺血的改善作用

背景:内皮祖细胞移植为肢体缺血的治疗提供了新的选择。目的:研究人外周血来源内皮祖细胞移植对改善肢体缺血的作用。方法:采用Ficoll密度梯度离心法获取人外周血单个核细胞,体外诱导扩增6d后,检测其内皮祖细胞特异性标志的表达,并将荧光染料标记后的贴壁细胞通过缺血局部多点注射移植到后肢缺血的裸鼠动物模型体内,以评价其治疗效果。结果与结论:从人外周血单个核细胞诱导出的贴壁细胞可表达内皮祖细胞特异性标志物CD133、CD34和血管内皮生长因子受体2,说明从人外周血单个核细胞中可诱导出内皮祖细胞。移植内皮祖细胞后裸鼠缺血后肢的坏死情况和毛细血管密度均明显改善(P<0.05);在缺血后肢肌肉石蜡切片中可见分散不均的红色和黄绿色荧光标记的内皮祖细胞的掺入。表明移植的内皮祖细胞可以定向整合到缺血局部,改善裸鼠的后肢缺血。     

PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.     

Diabetic impairments in NO-mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha

Endothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for the diabetic defect in circulating and wound EPCs. Since mobilization of BM EPCs occurs via eNOS activation, we hypothesized that eNOS activation is impaired in diabetes, which results in reduced EPC mobilization. Since hyperoxia activates NOS in other tissues, we investigated whether hyperoxia restores EPC mobilization in diabetic mice through BM NOS activation. Additionally, we studied the hypothesis that impaired EPC homing in diabetes is due to decreased wound level stromal cell-derived factor-1alpha (SDF-1alpha), a chemokine that mediates EPC recruitment in ischemia. Diabetic mice showed impaired phosphorylation of BM eNOS, decreased circulating EPCs, and diminished SDF-1alpha expression in cutaneous wounds. Hyperoxia increased BM NO and circulating EPCs, effects inhibited by the NOS inhibitor N-nitro-L-arginine-methyl ester. Administration of SDF-1alpha into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, and wound healing. Thus, hyperoxia reversed the diabetic defect in EPC mobilization, and SDF-1alpha reversed the diabetic defect in EPC homing. The targets identified, which we believe to be novel, can significantly advance the field of diabetic wound healing.     

脐血内皮祖细胞尾静脉与局部注射治疗糖尿病下肢缺血

背景:内皮祖细胞治疗糖尿病下肢缺血临床及动物实验多采用局部肌肉注射。目的:比较脐血内皮祖细胞鼠尾静脉与局部注射治疗糖尿病下肢缺血效果的差异。方法:取Wistar雄性大鼠分成5组:①糖尿病射线照射后结扎双后肢股动脉,尾静脉注射内皮祖细胞(DLV)。②糖尿病结扎双后肢股动脉左后肢局部肌肉注射PBS(DLC),右后肢局部肌肉注射内皮祖细胞(DLM)。③正常大鼠射线照射后结扎双后肢,尾静脉注射内皮祖细胞(NLV)。④糖尿病不结扎不注射内皮祖细胞(DC)。用绿色荧光示踪内皮祖细胞,苏木精-伊红染色检测肌纤维间毛细血管数,RT-PCR检测双后肢肌肉血管内皮生长因子mRNA水平。结果与结论:DLV组与DLM组比较,右后肢腓肠肌溃疡及缺血好转明显,二者无明显区别;有明显荧光,差别不明显,Ⅷ因子免疫组织化学染色肌纤维间毛细血管数多,相互间无明显差别;腓肠肌血管内皮生长因子表达差异无显著性意义(P>0.05)。提示脐血内皮祖细胞治疗糖尿病大鼠下肢缺血尾静脉注射与局部肌肉注射效果相当。     

运动训练动员骨髓内皮祖细胞促进缺血下肢血管新生的研究

目的探讨运动训练对骨髓内皮祖细胞(EPCs)的动员及对缺血下肢微血管生成的影响。方法 SD大鼠60只随机平均分为A、B、C三组,A、B组建立大鼠下肢缺血模型,C组仅作皮肤切开缝合。建模1周后A组大鼠跑步训练(30 min/d)。B、C组日常活动,1周后通过细胞培养,观察外周血EPCs数量的变化,4周后检测缺血组织块免疫组化微血管密度(MVD)的表达。结果 A组外周血培养的EPCs平均为(14.18±2.60)个/HPF,显著高于B组为5.59±1.34/HPF,P<0.01;A组MVD:39.94±4.01/HPF,显著高于B组(27.23±3.64)个/HPF,P<0.01;B组的外周血EPCs、组织MVD均明显高于C组(P<0.01),A、B、C 3组之间存在显著性差异(P<0.01)。结论运动训练能动员骨髓EPC,促进缺血肢体血管形成,改善局部血供。     

脐血内皮祖细胞尾静脉与局部注射治疗糖尿病下肢缺血

背景：内皮祖细胞治疗糖尿病下肢缺血临床及动物实验多采用局部肌肉注射。目的：比较脐血内皮祖细胞鼠尾静脉与局部注射治疗糖尿病下肢缺血效果的差异。方法：取Wistar雄性大鼠分成5组：①糖尿病射线照射后结扎双后肢股动脉,尾静脉注射内皮祖细胞（DLV）。②糖尿病结扎双后肢股动脉左后肢局部肌肉注射PBS（DLC）,右后肢局部肌肉注射内皮祖细胞（DLM）。③正常大鼠射线照射后结扎双后肢,尾静脉注射内皮祖细胞（NLV）。④糖尿病不结扎不注射内皮祖细胞（DC）。用绿色荧光示踪内皮祖细胞,苏木精-伊红染色检测肌纤维间毛细血管数,RT-PCR检测双后肢肌肉血管内皮生长因子mRNA水平。结果与结论：DLV组与DLM组比较,右后肢腓肠肌溃疡及缺血好转明显,二者无明显区别;有明显荧光,差别不明显,Ⅷ因子免疫组织化学染色肌纤维间毛细血管数多,相互间无明显差别;腓肠肌血管内皮生长因子表达差异无显著性意义（P〉0.05）。提示脐血内皮祖细胞治疗糖尿病大鼠下肢缺血尾静脉注射与局部肌肉注射效果相当。     

Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing

BM-derived endothelial progenitor cells (EPCs) are critical and essential for neovascularization in tissue repair and tumorigenesis. EPCs migrate from BM to tissues via the bloodstream, but specific chemotactic cues have not been identified. Here we show in mice that the absence of CCR5 reduced vascular EPC accumulation and neovascularization, but not macrophage recruitment, and eventually delayed healing in wounded skin. When transferred into Ccr5-/- mice, Ccr5+/+ BM cells, but not Ccr5-/- cells, accumulated in the wound site, were incorporated into the vasculature, and restored normal neovascularization. Consistent with these observations, CCL5 induced in vitro EPC migration in a CCR5-dependent manner. Moreover, expression of VEGF and TGF-β was substantially diminished at wound sites in Ccr5-/- mice, which suggests that EPCs are important not only as the progenitors of endothelial cells, but also as the source of growth factors during tissue repair. Taken together, these data identify the CCL5/CCR5 interaction as what we believe to be a novel molecular target for modulation of neovascularization and eventual tissue repair.     

Cardiovascular disease: potential impact of stem cell therapy

Atherosclerotic vascular disease becomes a clinical problem when there is sufficient atherosclerotic plaque burden and/or endothelial dysfunction to cause a limitation of nutrient blood flow to tissues. However, once myocardial infarction has occurred, there is little, if any, way to stimulate the growth of new blood vessels or cardiac muscle to replace that which has been lost. The potential use of hematopoietic stem cells (HSCs) to treat cardiovascular disease has recently been suggested from preclinical and clinical studies. HSCs are precursors of all the blood cells, but they may also give rise to cells of the vascular system, endothelial cells in the form of endothelial progenitor cells (EPCs). Clinical trials have been conducted in patients with either acute myocardial infarction or limb ischemia to determine the initial effectiveness and safety of this treatment approach. These studies demonstrated the potential clinical effectiveness of this stem cell approach to the treatment of patients with acute myocardial ischemia and limb ischemia. Today, more preclinical studies are planned to elucidate the mechanism by which transplanted stem cells can home and differentiate into these endothelial cells and cardiac muscle cells. At the same time, new clinical trials are planned to evaluate both chronic, stable as well as acute myocardial ischemia and limb ischemia with CD34⁺ and CD133⁺ stem cells, as well as with further selected EPCs and mesenchymal stem cells.     

Therapeutic angiogenesis by autologous cell implantation

Therapeutic angiogenesis is an effective means for tissue salvage in patients with critical limb ischemia. Angiogenesis is defined as a formation of new blood vessels by sprouting of preexisting mature endothelial cells(ECs). In contrast, vasculogenesis is referred to as the creation of primordial blood vessels from endothelial progenitor cells (EPCs) or angioblasts. Neovascular formation in adults has been considered to result exclusively from the former process(i.e., angiogenesis). However, we and other researchers recently identified EPCs in human peripheral blood(PB), and circulating EPCs have been shown to accumulate at active angiogenic sites and to participate in neovascularization, a notion consistent with 'postnatal vasculogenesis'. EPCs in adults originate from bone marrow(BM), and we recently have demonstrated that in vivo implantation of autologous BM-MNCs effectively augmented ischemia--induced neovascularization in animal studies as well as human trial(TACT Trial). Here we summarize recent advances in cell transplantation-mediated therapeutic angiogenesis.     

Angiogenic cell therapy

Therapeutic angiogenesis is an effective means for tissue salvage in patients with critical limb ischemia. Angiogenesis is defined as a formation of new blood vessels by sprouting of preexisting mature endothelial cells(ECs). In contrast, vasculogenesis is referred to as the creation of primordial blood vessels from endothelial progenitor cells (EPCs) or angioblasts. Neovascular formation in adults has been considered to result exclusively from the former process (i.e., angiogenesis). However, we and other researchers recently identified EPCs in human peripheral blood(PB), and circulating EPCs have been shown to accumulate at active angiogenic sites and to participate in neovascularization, a notion consistent with 'postnatal vasculogenesis'. EPCs in adults originate from bone marrow (BM), and we recently have demonstrated that in vivo implantation of autologous BM-MNCs effectively augmented ischemia-induced neovascularization in animal studies as well as human trial(TACT Trial). Here we summarize recent advances in cell transplantation-mediated therapeutic angiogenesis.     

Vascular endothelium regeneration therapy

CD34 positive cells were first defined as endothelial progenitor cells(EPCs) from circulating mononuclear cells in peripheral blood. EPCs have shown to be mobilized from bone marrow by the various factors, incorporate into sites of physiological and pathological neovascularization and differentiate into mature endothelial cells (ECs). Post-natal vasculogenesis has been considered to be involved in neovascularization of adult tissues. Recently, freshly isolated CD34 positive cells transplantation has started as clinical trial for ischemic diseases. In the clinical situation, we should consider the cell number and cell quality derived from the patients who have atherosclerosis background, especially diabetes. Ex vivo expansion or gene modification of EPCs could be the strategies for the next generation cell therapy to overcome these issues.     

Bone marrow-derived endothelial progenitor cells for vascular regeneration

It has recently been demonstrated that postnatal neovascularization is not restricted to angiogenesis, but also includes vasculogenesis. During adult vasculogenesis, bone marrow (BM)-derived endothelial progenitor cells (EPCs) are recruited to the systemic circulation in response to certain cytokines and/or tissue ischemia, and incorporate into sites of neovascularization. EPCs have also been investigated as therapeutic agents in a 'supply-side' approach to promoting neovascularization under pathological conditions. This review highlights the discovery of BM-derived EPCs and their therapeutic potential for vascular regeneration.     

Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2

Summary.  Background and objective:  Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. Methods and results:  We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP-2 and αvβ3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of αvβ3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. Conclusions:  HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction.     

Newly emerging concepts in blood vessel growth: recent discovery of endothelial progenitor cells and their function in tissue regeneration.

It has recently been established that bone marrow-derived endothelial progenitor cells (EPCs) are recruited to the systemic circulation and, in response to various cytokines, pharmacologic agents, and/or tissue ischemia, incorporate into sites of new blood vessel growth (neovascularization). These findings have changed our understanding of adult neovascularization by demonstrating that both preexisting endothelial cells and EPCs contribute to blood vessel formation during adult life. The following review article highlights the discovery of EPCs, their relationship to various clinical diseases, and their therapeutic potential for augmenting blood vessel formation.     

动员后外周血中CD34+细胞去除前后对改善肢体缺血疗效的研究

目的对动员的外周血单个核细胞（PBMNC）和动员的去除CD34^＋细胞的PBMNC移植治疗裸鼠下肢缺血的疗效进行比较，以探讨PBMNC的治疗机制。方法经过单采获得G-CSF动员的PBMNC后，一部分通过CD34磁珠抗体分选得到去除CD34^＋细胞的PBMNC。动员的PBMNC和动员的去除CD34^＋细胞的PBMNC荧光标记后按1×10^6细胞或相应体积的PBS分别局部肌肉注射移植到裸鼠缺血下肢。观察下肢血流灌注以及毛细血管密度。用ELISA法检测下肢肌肉的血管内皮生长因子（VEGF）表达，并进一步观察表达的VEGF是否由移植细胞分泌。结果PBMNC移植后缺血下肢血流明显恢复，毛细血管密度明显增加，但去除CD34^＋细胞的PBMNC移植组疗效有所下降。细胞移植后4周，PBMNC组的血流灌注由（20．3±4．2）％恢复为（96．4±5．6）％，对照组仅恢复为（71．3±4．4）％（P〈0．01），去除CD34^＋细胞组恢复为（83．8±5．2）％（P〈0．05）。PBMNC组血管密度为（521±47）／mm^2，去除CD34^＋细胞组为（3964-21）／mm^2（P〈0．05），但仍高于对照组[（276±43）／mm^2]（P〈0．05）。在PBMNC组可以观察到移植的细胞整合到缺血的毛细血管壁。缺血肌肉VEGF的表达明显升高，其共表达VEGF和移植的单个核细胞。结论移植G-CSF动员的PBMNC不但可以通过干细胞整合到血管壁的机制促进血管生长，还可以通过提供细胞因子的机制促进血管生长。去除CD34^＋细胞削弱了动员的PBMNC移植治疗肢体缺血的血管新生效应。     

自体外周血干细胞移植治疗糖尿病下肢缺血效果观察

目的 探讨自体外周血干细胞(PBSC)移植治疗糖尿病性下肢缺血的疗效。方法 应用自体PBSS移植治疗18例30条糖尿病性下肢缺血。采用主观、客观评价指标对疗效进行观察评定。结果 自体PBSC移植后1个月患者疼痛、冷感、麻木症状明显改善,改善率分别为96.7％ (29/30)、100％(30/30)、95.8％ (23/24);间歇性跛行明显缓解,总有效率为76.9％ (10/13)。3个月后踝肱指数(ABI)升高,由术前的0.60±0.11增加到术后的0.71±0.12,差异有统计学意义(t=-6.882,P＜0.01)。93.3％(28/30)的患者皮氧分压不同程度地升高;同时患者的足部感染得到控制,溃疡或足趾坏疽好转或愈合。移植后所有患者均未出现并发症和明显不良反应。结论 自体PBSC移植治疗糖尿病性下肢缺血是一种相对简单、安全、有效的方法。