Mobilizing endothelial progenitor cells

Mobilization of endogenous endothelial progenitor cells (EPCs) from the bone marrow may be an alternative way to increase neovascularization and may be used as therapeutic option for the treatment of ischemic cardiovascular diseases. In this review, we discuss the EPC mobilizing effects of pro-inflammatory cytokines such as granolocyte monocyte colony-stimulating factor and granulocyte colony-stimulating factor, growth factors such as vascular endothelial growth factor, placental growth factor, erythropoietin, and angiopoietin-1, chemokines such as stromal cell-derived factor-1, hormones such as estrogens and lipid-lowering and anti-diabetic drugs, as well as physical activity.     

人和大鼠内皮祖细胞培养及其生长特性的比较

目的建立人内皮祖细胞（hEPCs）离体培养的方法,探讨培养条件,观察细胞生长状态、形态。xd同时行大鼠EPCs（rEPCs）培养。建立hEPCs和rEPCs培养体系,比较两者不同的生长条件和生长状态。方法：取人脐带血80-120ml/袋,先分离单个核细胞,后使用磁珠细胞分选法（MACS）,分选出CD133＋/VEGFR2＋的细胞,进行流式细胞检测,发现双阳性细胞占48.79%。用差速贴壁法培养5-9天,倒置相差显微镜观察细胞形态并照相、免疫荧光染色后荧光显微镜下观察照相。取大鼠骨髓,冲洗骨髓腔,离心沉淀出细胞,差速贴壁培养法培养、观察。结果：（1）hEPCs在普通光镜下呈索条状、卵圆形。（2）细胞吞噬DiI-acLDL、UEA染料后可在荧光显微镜下特异显色,证明细胞有吞噬功能,推断为hEPCs。（3）CD133＋/VEGFR2＋的hEPCs细胞,占使用MACS筛选后细胞比例为48.79%。（4）从骨髓中分离出的rEPCs生长活力明显优于hEPCs。结论：（1）该方法培养的hEPCs和rEPCs生长活性好,在普通光镜下呈索条状、卵圆形或铺路石样;（2）hEPCs在细胞数量上可不少于rEPCs,从骨髓中培养出的rEPCs增殖力优于脐血来源的hEPCs。此实验比较并完善了两种不同来源内皮祖细胞培养的方法学及其生长特征,有利于根据不同的细胞培养特性来选择应用于内皮祖细胞的实验研究。     

Erythropoietin regulates endothelial progenitor cells

Circulating bone marrow-derived endothelial progenitor cells (EPCs) promote vascular reparative processes and neoangiogenesis, and their number in peripheral blood correlates with endothelial function and cardiovascular risk. We tested the hypothesis that the cytokine erythropoietin (EPO) stimulates EPCs in humans. We studied 11 patients with renal anemia and 4 healthy subjects who received standard doses of recombinant human EPO (rhEPO). Treatment with rhEPO caused a significant mobilization of CD34(+)/CD45(+) circulating progenitor cells in peripheral blood (measured by flow cytometry), and increased the number of functionally active EPCs (measured by in vitro assay) in patients (week 2, 312% +/- 31%; week 8, 308% +/- 40%; both P <.01 versus baseline) as well as in healthy subjects (week 8, 194% +/- 15%; P <.05 versus baseline). The effect on EPCs was already observed with an rhEPO dose of about 30 IU/kg per week. Administration of rhEPO increased the number of functionally active EPCs by differentiation in vitro in a dose-dependent manner, assessed in cell culture and by tube formation assay. Furthermore, rhEPO activates the Akt protein kinase pathway in EPCs. Erythropoietin increases the number of functionally active EPCs in humans. Administration of rhEPO or EPO analogs may open new therapeutic strategies in regenerative cardiovascular medicine.     

Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells

Circulating endothelial progenitor cells (EPC) are incorporated into newly formed capillaries, enhance neovascularization after hind limb ischemia and improve cardiac function after ischemic injury. Incorporated progenitor cells may also promote neovascularization and cardiac regeneration by releasing factors, which act in a paracrine manner to support local angiogenesis and mobilize tissue residing progenitor cells. Therefore, we analyzed the expression profile of cytokines in human peripheral blood-derived EPC as opposed to human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), and CD14(+) monocytes by microarray technology. A gene tree analysis revealed a distinct expression pattern of angiogenic growth factors in EPC, mature endothelial cells, and CD14(+) monocytes. VEGF-A, VEGF-B, SDF-1, and IGF-1 mRNA levels were higher in EPC as compared to HUVEC or HMVEC. The enhanced mRNA expression was paralleled by a significant release of VEGF, SDF-1, and IGF-1 protein into the cell culture supernatant of EPC. Moreover, immunohistological analysis of ischemic limbs from nude rats revealed that VEGF is also released from recruited human EPC in vivo. As a functional consequence, conditioned medium of EPC induced a strong migratory response of mature endothelial cells, which was significantly inhibited by VEGF and SDF-1 neutralizing antibodies. Finally, conditioned medium of EPC significantly stimulated the migration of cardiac resident c-kit(+) progenitor cells in vitro. Taken together, EPC exhibit a high expression of angiogenic growth factors, which enhanced migration of mature endothelial cells and tissue resident cardiac progenitor cells. In addition to the physical contribution of EPC to newly formed vessels, the enhanced expression of cytokines may be a supportive mechanism to improve blood vessel formation and cardiac regeneration after cell therapy.     

Platelet-induced differentiation of endothelial progenitor cells

Endothelial progenitor cells (EPCs) have the potential to home at sites of vascular lesions and to contribute to revascularization. This homing is a highly concerted mechanism, which involves chemotaxis, adhesion, migration, and finally integration of the cells into the target tissue. Only recently has the platelet been identified as a central mediator of EPC homing. Adherent platelets were able to mediate chemotaxis and adhesion of EPCs, a process that involved P-selectin glycoprotein ligand 1 and very late antigen-4 (VLA-4). Recent studies suggest that platelet-derived stromal cell-derived factor-1 is also involved centrally in the recruitment of EPCs. Furthermore, platelets induce progenitor cell migration by platelet-derived growth factor AB. Recent in vivo data confirm the recruitment of EPCs to sites of vascular lesions after vessel denudation by activated platelets and fibrin. Moreover, when coincubated with platelets, EPCs differentiate to mature endothelial cells and have the potential to migrate and colonize a platelet thrombus. The described interaction of EPCs with platelets represents a novel mechanism of vascular remodeling and healing of endothelial lesions.     

有关内皮祖细胞的研究进展

内皮细胞起着维持人体血管正常功能的重要作用,其功能紊乱及损伤贯穿心脑血管疾病发生发展的各环节.由于其增殖能力弱、不能远距离迁移等缺陷限制了它在血管疾病治疗方面的应用.内皮祖细胞(endothelial progenitor cells, EPCs)的发现正弥补了这一欠缺.1997年Asahara等[1]首先由外周血中分离出了骨髓来源的CD34+细胞和KDR+细胞,即现在所说的EPCs,是ECs的前体细胞,它具有缺血区定向归巢并分化为成熟内皮细胞、迟发高增殖潜能等特性,不仅参与胚胎发育时的血管发生,在出生后也起着促进受损内皮修复、微血管新生等重要作用.本文就EPCs的来源及其表面抗原、影响EPCs数目及功能的因素、EPCs的主要临床应用综述如下.     

Vascular repair by endothelial progenitor cells

Accumulating evidence indicates the impact of endothelial progenitor cells (EPCs) in vascular repair. In patients, the number of EPCs is negatively correlated with the severity of atherosclerosis. In various animal models, transplantation of bone marrow-derived progenitor cells could sufficiently rescue organ function and enhance vascular repair and tissue regeneration. Increase in the number of circulating progenitors, induced by cell transfusion or enhanced mobilization, can also enhance restoration and integrity of the endothelial lining, suppress neointimal formation, and increase blood flow to ischaemic sites. However, the beneficial outcome of EPC infusion very much depends on the growth and differentiation factors within the tissue, cell-to-cell interactions, and the degree of injury. As highlighted by several studies, EPCs derive from different sources including bone marrow and non-bone marrow organs such as the spleen, the functional repair properties of which may vary with the maturation state of the cell. Thus, understanding the molecular mechanisms involved in EPC-repairing processes is essential. In the present review we focus on the role of EPCs in vascular diseases, and we provide an update on the mechanisms of EPC mobilization, homing, and differentiation.     

骨髓源性内皮祖细胞与内皮细胞共培养促进其向成熟内皮细胞分化的研究

目的：探讨共培养的内皮细胞对内皮祖细胞向成熟内皮细胞分化的影响。方法：分离1～2个月龄,SD大鼠股骨髓,个核细胞（MNCs）,MNCs,Dil-ac-LDL与FITC-UEA-1荧光双染鉴定内皮细胞的特性。应用贴块法培养大鼠腹主动脉的内皮细胞,vWF免疫组化染色进行鉴定。采用Transwell培养板,上、下室分别加入内皮祖细胞和内皮细胞,15%胎牛血清的NO.2 DMEM/F12培养液培养14 d,倒置相差显微镜观察培养内皮祖细胞的形态。RT-PCR检测eNOS、vWF mRNA,流式细胞术检测CD31及KDR的表达。结果：荧光双染显示培养的单个核细胞具有内皮祖细胞特性;共培养的内皮祖细胞呈短梭型、铺路石状,培养至4 w时有复杂的网状结构形成。RT-PCR检测显示,eNOS及vWF mRNA表达均较对照组显著增加（P〈0.05）。流式细胞术分析表明,共培养组的内皮祖细胞CD31及KDR的表达,也显著高于对照组（分别为P〈0.05及P〈0.01）。结论：与内皮细胞共培养可促进内皮祖细胞向成熟内皮细胞分化。     

内皮祖细胞功能状态及其调节

自1997年Asahara等首次在成体外周血中发现血管内皮祖细胞（EPC）以来,有关EPC的研究口益增多,彻底改变了传统血管新生的概念。同时,大量动物实验和初步临床研究均显示EPC在治疗缺血性心脏病、外周血管缺血性疾病及血管损伤后再狭窄等方面具有广阔的临床应用前景。近年来,越来越多的学者倾向于EPC是一种细胞的功能状态,而非特指某一群细胞实体。本文围绕这一观点及EPC功能状态的调节作一综述。     

Nifedipine improves endothelial function: role of endothelial progenitor cells

Nifedipine has been shown to improve endothelial function. Recent studies have indicated that endothelial function is correlated with the number of circulating endothelial progenitor cells (EPCs), but it is unclear whether nifedipine affects the number and function of EPCs. The aims of this study were to determine the effects of nifedipine on the number and function of EPCs and to investigate the relationship between improvement of endothelial function and EPC numbers in patients with hypertension. Stage 1 hypertensive men (n=37) were randomly divided into the nifedipine group and the control untreated group. The nifedipine group was administered slow-release nifedipine (20 mg) once daily. At baseline and after 4 weeks, flow-mediated dilation, blood pressure, biochemical data, and number of circulating CD34+CD133+ progenitor cells and EPCs were measured. The direct effects of nifedipine on EPC number and function were assessed in vitro. In the nifedipine group, flow-mediated dilation and the numbers of circulating CD34+CD133+ progenitor cells and EPCs were increased, along with a decrease of serum malondialdehyde low-density lipoprotein. The improvement of flow-mediated dilation by nifedipine was correlated with the increase of circulating CD34+CD133+ progenitor cells. Nifedipine also improved angiogenesis-related functions of EPCs (differentiation, migration, and resistance to oxidative stress) in vitro. Thus, nifedipine improved endothelial function and EPC function in stage 1 hypertensive subjects. The latter action may be mediated by reduction of oxidative stress and suppression of EPC apoptosis. These results demonstrate that nifedipine preserves endothelial integrity in patients with hypertension, at least partly, by enhancing EPC numbers and activity.     

Circulating endothelial progenitor cells during human pregnancy

The precise molecular and cellular mechanisms that regulate maternal vascular development during gestation are largely unknown. Endothelial progenitor cells (EPCs), which play an important role in vascular homeostasis, have been discovered in the circulation. We examined the level of circulating EPCs throughout uncomplicated pregnancies (n = 20) and assessed the correlation between serum estradiol levels and the number of EPCs. The number of circulating EPCs increased gradually and paralleled the progression of gestational age. In addition, the number of EPCs correlated significantly with the level of serum estradiol. The present study suggests that EPCs may play an important role in the regulation and maintenance of the placental development and vascular integrity during pregnancy.     

Cellular biomarkers of endothelial health: microparticles,endothelial progenitor cells,and circulating endothelial cells

Endothelial dysfunction, the shift from a healthy endothelium to a damaged pro-coagulative, pro-inflammatory, and pro-vasoconstrictive phenotype, is an early event in many chronic diseases that frequently precedes cardiovascular complications. Functional assessment of the endothelium can identify endothelial damage and predict cardiovascular risk; however, this assessment provides little information as to the mechanisms underlying development of endothelial dysfunction. Changes in plasma asymmetric dimethyl arginine levels, markers of lipid peroxidation, circulating levels of inflammatory mediators, indices of coagulation and cellular surrogates such as microparticles, circulating endothelial cells, and endothelial progenitor cells may reflect alterations in endothelial status and as such have been defined as “biomarkers” of endothelial function. Biomarkers may be chemical or cellular. This review examines some markers of endothelial dysfunction, with a particular focus on cellular biomarkers of endothelial dysfunction and their diagnostic potential.     

Beneficial effects of statins on endothelial progenitor cells

ABSTRACT:: In recent years, endothelial progenitor cells (EPCs) have been demonstrated to play an important role during tissue vascularization and endothelium homeostasis in adults. In addition, EPCs have been implicated in the pathophysiology of cardiovascular and cerebrovascular disease, such that a decreased number of EPCs may not only be a risk indicator but also a potential therapeutic target. Of the many agents that have been examined to increase EPCs and enhance their function, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or statins are one of the most intriguing. Accumulated evidence has demonstrated that statins promote EPC mobilization, proliferation, migration, adhesion, differentiation and reduce senescence and apoptosis independent of their serum lipid-lowering effect. This review summarizes the understanding of current mechanisms explaining the myriad of beneficial effects of statins on EPCs and discusses future challenges for studies involving statins and subpopulations of EPCs. However, the pharmacologic mechanisms of action of statins on EPCs remain at the cellular level, whereas the putative molecular mechanisms await further studies.     

Material-based deployment enhances efficacy of endothelial progenitor cells

Cell-based therapies are attractive for revascularizing and regenerating tissues and organs, but clinical trials of endothelial progenitor cell transplantation have not resulted in consistent benefit. We propose a different approach in which a material delivery system is used to create a depot of vascular progenitor cells in vivo that exit over time to repopulate the damaged tissue and participate in regeneration of a vascular network. Microenvironmental conditions sufficient to maintain the viability and outward migration of outgrowth endothelial cells (OECs) have been delineated, and a material incorporating these signals improved engraftment of transplanted cells in ischemic murine hindlimb musculature, and increased blood vessel densities from 260 to 670 vessels per mm², compared with direct cell injection. Further, material deployment dramatically improved the efficacy of these cells in salvaging ischemic murine limbs, whereas bolus OEC delivery was ineffective in preventing toe necrosis and foot loss. Finally, material deployment of a combination of OECs with another cell population commonly isolated from peripheral or cord blood, endothelial progenitor cells (EPCs) returned perfusion to normal levels in 40 days, and prevented toe and foot necrosis. Direct injection of an EPC/OEC combination was minimally effective in improving limb perfusion, and untreated limbs underwent autoamputation in 3 days. These results demonstrate that vascular progenitor cell utility is highly dependent on the mode of delivery, and suggest that one can create new vascular beds for a variety of applications with this material-controlled deployment of cells.