细胞介导的治疗性血管发生与缺血性心脏病的治疗

干细胞介导的治疗性血管发生正逐渐成为血管再生研究领域新的热点,内皮祖细胞在其中起着重要作用。现对内皮祖细胞在缺血性心脏病治疗性血管发生方面的作用机制及其研究进展作一综述。     

内皮前体细胞移植:冠心病治疗的新策略

干细胞研究的进展和基因治疗技术在心血管的应用为缺血性心脏病的治疗提供了新策略。基因修饰后内皮前体细胞移植既补充了功能不良且数量不足的内皮前体细胞又诱导并促进了局部新血管的生成 ,展示了美好的临床应用前景。本文回顾了近几年来缺血性心脏病治疗的新概念 ,重点介绍了国外内皮前体细胞及其基因修饰应用的新进展 ,并对存在的问题和应用前景分别进行了讨论和展望     

2型糖尿病患者外周血内皮祖细胞数量和功能的变化

目的观察2型糖尿病（DM）患者外周血内皮祖细胞（EPCs）数量和功能的改变。方法选择2型DM患者16例和对照组19例，密度梯度离心法收集外周血单个核细胞（MNCs），诱导分化培养7d后，荧光染色和流式细胞术分别鉴定贴壁细胞为EPCs。采用二苯基四氮唑嗅盐（MTT）比色法、黏附能力测定实验和体外血管生成实验检测EPCs的增殖能力、黏附能力和体外血管生成能力。结果2型DM患者外周血EPCs数量明显减少[（3．1±1．2）×10^5]：[（3．9±1．1）×10^5]，P〈0．05。且2型DM患者外周血EPCs黏附能力[（50±15）：（60±11）细膨×200视野，P〈0．05]，增殖能力[（0．170±0．056）：（0．225±0．071）OD值，P〈0．05]，体外血管生成能力均明显受损。结论2型DM患者外周血EPCs的数量减少，且其增殖、黏附和血管生成能力受损。     

糖尿病引起内皮祖细胞损伤机制研究进展

内皮祖细胞来源于骨髓细胞,在维持内皮细胞完整性和缺血组织的血管新生方面起关键作用.糖尿病可引起内皮祖细胞功能障碍,其机制包括促进活性氧簇产生,降低内皮型一氧化氮合酶活性,刺激炎性反应,抑制与血管新生有关的蛋白信号通路等,最终导致血管新生障碍,这是糖尿病患者出现心血管并发症、伤口愈合延迟甚至面临截肢危险的重要原因.探索作用于受损糖尿病内皮祖细胞功能的新靶点,可通过改善内皮祖细胞功能,开拓治疗糖尿病心血管病变和足病的新途径.     

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.     

Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia–reperfusion injury in rats

Aims/hypothesis A reduction in the number of endothelial progenitor cells (EPCs) is considered a plausible cause of increased cardiovascular risk in diabetes mellitus. The aim of this study was to test the hypothesis that weak bone marrow mobilisation is responsible for the decrease in circulating EPCs in diabetes.Materials and methods We employed a model of hindlimb ischaemia–reperfusion (I/R) injury to study mobilisation of EPCs in control and streptozotocin diabetic rats. EPCs were defined by flow cytometry as Sca-1⁺ and Sca-1⁺c-kit⁺ peripheral blood cells and further characterised by the expression of CD31, von Willebrand factor and fetal liver kinase-1. Capillary density was evaluated by immunofluorescent staining of vWF. We also determined plasma levels of stromal cell-derived factor (SDF-1) and vascular endothelial growth factor (VEGF) by ELISA and muscle expression of hypoxia-induced factor (HIF-1α) by Western blotting.Results In control rats, EPCs showed a mobilisation curve within 7 days, while diabetic rats were completely unable to mobilise EPCs after I/R injury. As a consequence, diabetic rats showed no compensatory increase in muscle capillary density. Defective EPC mobilisation in diabetes was associated with altered release of SDF-1 and VEGF and inability to upregulate muscle HIF-1α. Both insulin administration and premedication with granulocyte-colony stimulating factor and stem cell factor led to partial recovery in post-ischaemic mobilisation of EPCs in diabetic rats.Conclusions/interpretation Defective ischaemia-induced bone marrow mobilisation of EPCs impedes compensatory angiogenesis in ischaemic tissues of diabetic animals. Growth factor administration together with blood glucose control may offer a rational therapeutic strategy for diabetic ischaemic syndromes.     

Role of endothelial progenitors and other bone marrow-derived cells in the development of the tumor vasculature

Increasing evidence suggests the importance of bone marrow-derived cells for blood vessel formation (neovascularization) in tumors, which can occur in two mechanisms: angiogenesis and vasculogenesis. Angiogenesis results from proliferation and sprouting of existing blood vessels close to the tumor, while vasculogenesis is believed to arise from recruitment of circulating cells, largely derived from the bone marrow, and de novo clonal formation of blood vessels from these cells. Although bone marrow-derived cells are crucial for neovascularization, current evidence suggests a promotional role of these cells on the existing blood vessels rather than de novo neovascularization in tumors. This is believed to be due to the highly proangiogenic features of these cells. The bone marrow-derived cells are heterogeneous, consisting of many different cell types including endothelial progenitor cells, myeloid cells, lymphocytes, and mesenchymal cells. These cells are highly orchestrated under the influence of the specific tumor microenvironment, which varies depending on the tumor type, thereby tightly regulating neovascularization in the tumors. In this review, we highlight some of the recent findings on each of these cell types by outlining some of the essential proangiogenic cytokines that these cells secrete to promote tumor angiogenesis and vasculogenesis.     

Transfer of endothelial progenitor cells improves myocardial performance in rats with dilated cardiomyopathy induced following experimental myocarditis

Endothelial progenitor cells (EPCs) home to sites of tissue injury and differentiate into mature endothelial cells. Their transfer feasibility has been proven in models of hindlimb ischemia and myocardial infarction. We investigated, the effect of delivery of spleen-derived EPC in a rat model of inflammatory-mediated myocardial damage. Male Lewis rats (N=25) were immunized against myosin. Healthy donor Lewis rats were sacrificed, their spleens harvested, separated on Ficoll gradient centrifugation, and grown on fibronectin coated plates with endothelial cell medium for 5 days. Ten days after myosin immunization, spleen cell derived EPC were collected, and labeled 2 x 10(7) cells per rat were injected into the femoral vein of diseased rats. Cell transplantation was repeated twice, 2 and 4 weeks after initial cell transfer. Rats with inflammatory-mediated cardiomyopathy exhibited a significant mobilization of EPC from the bone marrow to the periphery and their ability to adhere to fibronectin, mature endothelial cells and cultured cardiomyocytes was significantly reduced when compared to healthy rats. Transfer of EPC resulted in a functional improvement in cardiac performance evident by higher fractional shortening by echocardiography (a 15% increase). Histological studies exhibited reduced scar tissue and thickened ventricular walls in rats receiving EPC as compared with untreated animals. EPC transfer is effective in attenuating myocardial damage in a model of non-ischemic dilated cardiomyopathy.     

低氧预刺激对大鼠骨髓源性内皮祖细胞生物学活性的影响

目的探讨低氧预刺激对内皮祖细胞(EPC)生物学活性的影响,以进一步提高EPC移植治疗的效能。方法2003-02~2004-02在沈阳军区总医院将体外分离培养大鼠骨髓来源的EPC,分别置于正常氧环境和低氧环境中培养,观察低氧对EPC增殖能力、移行能力、成血管能力的影响。结果低氧组EPC增殖活性增加,镜下每高倍视野(HP)贴壁细胞数明显多于对照组[(91·0±8·0)对(42·5±5·3),P<0·01]。低氧预刺激可增加EPC对血管内皮生长因子(VEGF)的趋化移行能力,移行细胞数明显多于对照组[(112·2±13·2/HP对(43·4±9·2)/HP,P<0·01],且最大移行距离较长[(542·0±42·7)μm对(282·0±28·6)μm,P<0·01]。低氧组体外成血管能力强于对照组,且其VEGF及胚胎肝激酶(Flk-1)的mRNA表达亦明显增高。结论低氧预刺激可多方面提高EPC生物学活性,包括增殖活性、移行能力及体外成血管功能,其改变与VEGF及Flk-1表达的增高呈一致性。     

内皮祖细胞在心血管疾病治疗中的作用

1997年,Asahara等[1]定义了从外周血中分离出来的内皮祖细胞（endothelial progenitor cells,EPCs）.EPCs在动员、迁移后亦能形成新血管,这一过程后来被称为“血管新生”.EPCs对血管新生和内皮修复的作用展现了其潜在的治疗价值,改善EPCs功能、EPCs移植、内皮捕获支架等治疗心血管疾病提示有广泛的前景. 1 EPCs概述 自从发现外周循环中的EPCs以来,研究者已经应用多种方法来识别和分离此类细胞群.然而,EPCs仍未有共识性的定义.EPCs包含着一组细胞,这些细胞在不同的发展阶段中存在,从成血管细胞到分化成熟的内皮细胞不等.     

The role of propranolol in the treatment of infantile hemangioma

IntroductionInfantile hemangioma (IH) is one of the most common childhood tumors. There are various medical or surgical therapeutic options, all with suboptimal results. Recently, the successful use of propranolol for involution of IH was described. We report the results of a single-center experience with this therapeutic option.ObjectiveTo prospectively assess the efficacy and safety of propranolol in children with infantile hemangioma.MethodsWe performed a prospective analysis of clinical data of all patients with IH referred to a pediatric cardiology center for baseline cardiovascular assessment prior to propranolol therapy. Propranolol was given at a starting dose of 1 mg/kg/day and titrated to a target dose of 2–3 mg/kg/day according to clinical response. Efficacy was assessed through a photograph-based severity scoring scale. Safety was assessed by collecting data regarding significant side effects.ResultsStarting in 2010, 30 patients (15 female) were referred for propranolol treatment of IH, at a median age of six months (1–63 months). The mean target propranolol dose was 2.8 mg/kg/day, with a mean duration of therapy of 12 months. All patients experienced significant reduction of IH size and volume. There were no side effects.ConclusionsIn our experience propranolol appears to be a useful and safe treatment option for severe or complicated IH, achieving a rapid and significant reduction in their size. No adverse effects were observed, although until larger clinical trials are completed, potential adverse events should be borne in mind and consultation with local specialists is recommended prior to initiating treatment.     

Cardiovascular drugs in the treatment of infantile hemangioma

Since the introduction of propranolol in the treatment of complicated infantile hemangiomas(IH) in 2008, other different beta-blockers, including timolol, acetabutolol, nadolol and atenolol, have been successfully used for the same purpose. Various hypotheses including vasoconstriction, inhibition of angiogenesis and the induction of apoptosis in proliferating endothelial cells have been advanced as the potential beta-blockerinduced effect on the accelerated IH involution, although the exact mechanism of action of beta-blockers remains unknown. This has generated an extraordinary interest in IH research and has led to the discovery of the role of the renin-angiotensin system(RAS) in the biology of IH, providing a plausible explanation for the beta-blocker induced effect on IH involution and the development of new potential indications for RAS drugs such as angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers in the treatment of IH. This review is focused on the current use of cardiovascular drugs in the treatment of IH.     

血管内皮祖细胞在治疗动脉粥样硬化中的作用

<正>1997年,Asahara等[1]首次发现人体外周血中存在能分化为血管内皮细胞的前体细胞,将其命名为血管内皮祖细胞(endothelial progenitor cells,EPCs)。随后,人们对EPCs进行了大量的研究。内皮祖细胞不仅参与胚胎期的血管发生,而且在成体的血管生长发育中发挥重要作用,对于创伤修     

Regulation of endothelial progenitor cells by prostaglandin E1 via inhibition of apoptosis

Bone marrow derived endothelial progenitor cells (EPC) improve endothelial function and neoangiogenesis. Prostaglandin E1 (PGE1) is used for the treatment of patients with peripheral artery disease (PAD). However, the molecular effects are only partially understood. Treatment of C57/Bl6 mice with PGE1, 10 microg/kg BW increased the number of circulating Sca-1/VEGFR-2 positive EPC in the blood compared to vehicle (122+/-7% and 119+/-6% after 10 and 20 days). EPC in the bone marrow were upregulated to 125+/-11% (10 days) and 142+/-15% (20 days). PGE1 increased DiLDL/Lectin positive spleen-derived EPC to 170+/-20% and 174+/-14% after 10 and 20 days. Treatment with PGE1 enhanced in-vivo neoangiogenesis by 2-fold (disk assay, 218+/-27%). PGE1 enhanced the SDF-1 induced migratory capacity per number of EPC to 140+/-11%, 146+/-22% and 160+/-16% after 10, 14 and 20 days. Greater migratory capacity was associated with upregulation of expression of telomere repeat-binding factor (TRF2). EPC of PGE1-treated mice were characterized by reduced apoptosis. Similarly, PGE1 prevented H(2)O(2)-induced apoptosis in cultured human EPC. The effect is mediated by PI3-kinase. The effects of PGE1 on EPC were completely prevented by co-treatment with the NO-inhibitor L-NAME, 50 mg kg(-1) p.o. Treatment with the prostaglandin I2 derivative iloprost (10 microg/kg BW, 20 days) did not alter EPC numbers or function. Physical exercise is the basis of the treatment of patients with PAD. Voluntary running increased EPC numbers in mice. Treatment with PGE1 resulted in an additional increase of Sca-1/VEGFR-2- and DiLDL/lectin positive EPC as well as migration. n=10-24 for all groups, all effects p<0.05. In summary, prostaglandin E1 increases the number of EPC in the blood and the bone marrow in mice. The effect is additive to physical exercise, depends on nitric oxide and is characterized by reduction of PI3-kinase mediated apoptosis. PGE1-mediated upregulation of EPC is associated with improved EPC function and enhanced angiogenesis.     

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma

AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.     

人体外周血来源成体内皮前体细胞在体外培养过程中的单核细胞和巨噬细胞功能表现

目的：研究成体内皮前体细胞生物学特性,尤其对该种细胞所表现的单核细胞和巨噬细胞特点进行探讨。方法：人体外周血来源的单个核细胞被接种于纤维粘连蛋白(Fibronectin)包被的培养皿中,在 EGM-2培养液中培养。描述贴壁细胞生长曲线,对细胞表型及功能特点进行检测。结果：部分单个核细胞变形成为长梭形细胞,但未表现出明显的增殖能力。这些细胞可以表达部分内皮系表面标志物及单核细胞标志物 CD14。在第4,14,28天进行的 Dil 标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)和印度墨汁双摄取实验显示这种细胞能够同时吞噬这两种物质,却没有类似于内皮细胞的体外成血管现象,从而说明这种细胞有类似于单核细胞和巨噬细胞的吞噬功能。结论：人体外周血单个核细胞来源成年内皮前体细胞在体外培养过程中表现典型的单核巨噬细胞功能,且无明显增殖能力。