Arterio-venous gradient of endothelial progenitor cells across renal artery stenosis

A role for circulating progenitor cells have been recently demonstrated in many pathological conditions. Endothelial progenitor cells (EPCs) localize at sites of ischemia and stimulate neovasculogenesis. This small study was carried out to assess whether selective blood sampling during renal angiography could demonstrate an arterio-venous gradient of EPCs in 5 patients with renal artery stenosis. Surprisingly, we found that EPCs were more abundant in venous than in arterial blood, suggesting that the kidney subjected to chronic ischemia may become a source rather than a target of EPCs.     

Tissue plasminogen activator enhances mobilization of endothelial progenitor cells and angiogenesis in murine limb ischemia

Background

It has been reported that plasmin induces migration of endothelial cells. We hypothesized that tissue plasminogen activator (tPA) enhanced endothelial progenitor cell (EPC) mobilization from bone marrow (BM) into the circulation and angiogenesis in murine critical limb ischemia (CLI).

Methods

Forty male B6 mice were randomly divided into group 1 (control), group 2 [control + recombinant tPA (intra-venous 4 mg/kg)], group 3 (CLI) and group 4 (CLI + tPA).

Results

The results demonstrated higher MMP-9 activity in BM and circulatory SDF-1α level in groups 2 and 3 than in group 1, and highest in group 4 (all p < 0.01) at 18 h after CLI. Compared with circulation, SDF-1α level in BM showed an opposite trend (all p < 0.03). The circulating EPC (C-kit/CD31, Sca-1/KDR, CXCR4/CD34) levels at 18 h after CLI were higher in group 2 than in groups 1 and 3, and highest in group 4 (all p < 0.03). EPC (C-kit/CD31, Sca-1/KDR) levels in BM were lower in group 1 than in groups 2 to 4 (all p < 0.03), whereas number of CXCR4/CD34-positive EPCs in BM did not differ among the four groups at 18 h after CLI. Protein expressions of SDF-1α, CXCR4, eNOS, and VEGF, numbers of CD31+, CXCR4+, SDF-1α+, and vWF + cells through immunofluorescent staining, numbers of small vessels (< 15 μm) and blood flow measured by Laser Doppler in an ischemic area were significantly higher in group 4 than in group 3 (all p < 0.005) at day 14 after CLI.

Conclusion

tPA treatment enhanced number of circulating EPCs, angiogenesis, and blood flow to ischemic tissue in a murine model of limb ischemia.     

血管内皮祖细胞与脑缺血的相互关系

血管内皮祖细胞（endothelial progenitor cells,EPCs）为血管内皮细胞的前体细胞,具有胚胎时“血岛”细胞的分化特点,故又称之为血管母细胞（angioblast）。它具有一定的增殖能力,在一定条件下能分化为具有成熟血管内皮细胞表型的细胞,可修复损伤血管内皮,参与新生血管的形成,改善组织细胞的供血状态。在脑血管病中,EPCs参与了脑缺血损伤后的血管修复,对脑缺血后的血管网络重建有重要的意义。但不同的脑血管病其外周血EPCs的数量、增殖、迁移能力不同,而EPCs的动员、迁移、增殖过程又受多种内源性因素的调节。     

脐血内皮祖细胞治疗糖尿病大鼠下肢缺血的实验研究

目的研究脐血内皮祖细胞(EPC)治疗糖尿病下肢缺血的有效性及机制,为临床治疗糖尿病足病(DF)提供理论依据。方法取产妇足月产脐血分离单核细胞,培养7d,流式细胞仪鉴定细胞,并计数活细胞数。将Wistar雄性大鼠分成3组:(1)糖尿病大鼠射线照射后结扎双后肢股动脉,尾静脉注射血管EPC(DE组)。(2)糖尿病大鼠结扎双后肢股动脉,尾静脉注射缓冲液(DP组)。(3)正常大鼠射线照射后结扎双后肢,尾静脉注射EPC(NC组)。用绿色荧光(GFP)示踪EPC,Ⅷ因子免疫组化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(VEGF)mRNA水平。结果 DE、NC组右后肢腓肠肌溃疡及缺血明显好转,有明显荧光,肌纤维间毛细血管数多,VEGF表达高,DP组好转不明显,无荧光,毛细血管数少,VEGF表达少,其差异有统计学意义(P<0.05)。结论脐血EPC治疗糖尿病大鼠下肢缺血有效。     

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.     

Augmented neovascularization with magnetized endothelial progenitor cells in rats with hind-limb ischemia

Augmenting neovascularization with the use of endothelial progenitor cells (EPCs) is a therapeutic option to rescue critical limb ischemia (CLI). However, the outcomes have been not so satisfactory. The detectable number of injected EPCs at the ischemic site is rather small. If EPCs accumulate more and stay longer there, neovascularization will be augmented. In this study, we tested whether external magnetic forces (EMFs) accumulated magnetized EPCs (Mag-EPCs) at the ischemic site and thereby augmented neovascularization. We cultured peripheral blood-derived mononuclear cells to obtain EPCs and generated Mag-EPCs by a magnetofection method with nanoparticles. Prussian-blue staining revealed magnetic nanoparticles incorporated into the cytoplasms and nuclei of Mag-EPCs. The survival rate of Mag-EPCs at day 9 of culture was 98.7%, indicating no cell toxicity of magnetic nanoparticles. EMFs augmented adhesion capacity of Mag-EPCs not only in the static but also in the flow condition in vitro, compared to without EMFs. The migration capacity of Mag-EPCs with EMFs was 160% more than EPCs or Mag-EPCs without them. After an intravenous injection of Mag-EPCs into the rat with hind-limb ischemia, the recovery of blood flow and capillary density in the ischemic limb were significantly more (p < 0.01) with EMFs than without them. EMFs augmented neovascularization capacity of Mag-EPCs compared to EPCs alone. This method could be a new therapeutic strategy for patients with CLI.     

Angiogenic function of prostacyclin biosynthesis in human endothelial progenitor cells

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI(2)) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI(2) biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-alpha. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI(2) analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI(2) synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator-activated receptor (PPAR)delta agonist GW501516 but not by the selective PGI(2) receptor agonist cicaprost. Downregulation of PPARdelta by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPARdelta siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI(2) synthase siRNA, COX-1 siRNA, or PPARdelta siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI(2)/PPARdelta pathway is an important mechanism underlying proangiogenic function of EPCs.     

Autologous transplantation of peripheral blood endothelial progenitor cells (CD34+) for therapeutic angiogenesis in patients with critical limb ischemia.

AIM: Intramuscular injection of endothelial progenitor cells (EPCs) may constitute an alternative treatment strategy for patients with critical limb ischemia (CLI). We performed transplantations of EPCs (CD34(+)) extracted from peripheral blood in patients with CLI. The objective of this report is to present the method and early results of intramuscular autologous peripheral blood CD34(+) cell transplantation in the ischemic limb. METHODS: CD34(+) cell transplantation was performed in 2 limbs of 2 patients with CLI, in cases in which it was not possible to perform surgical or percutaneous revascularization. The patients received a granulocyte colony-stimulating factor (G-CSF) prior to the treatment. CD34(+) cells were retrieved from peripheral blood and injected directly into the muscle of the ischemic limb. RESULTS: CD34(+) cells retrieved in patient 1 were 1 x 10(5)/ml and in patient 2 were 1.6 x 10(5)/ml. Transcutaneous oxygen pressure in the foot increased and clinical symptoms improved. Newly visible collateral blood vessels were directly documented by angiography. CONCLUSION: Satisfactory clinical improvement was achieved by using peripheral blood EPCs (CD34(+)) in the patients with CLI. No complications arose following the intramuscular administration of peripheral blood CD34(+) cells.     

CXCR4基因过表达HUCB-late EPCs移植对大鼠后肢缺血组织血管新生的影响

目的观察转染rAAV_6-CXCR4和rAAV_6-GFP后,初步探索其是否对HUCB-late内皮组细胞(EPCs)的体外成血管能力及体内缺血组织血管新生有影响。方法用rAAV_6-CXCR4、rAAV_6-GFP感染HUCB-late EPCs,Western Blot检测CXCR4蛋白表达水平;应用基质胶成血管实验检测转染后细胞的体外成血管能力。构建SD大鼠后肢缺血模型,以一侧股动脉及分支结扎、离断的方法构建后肢缺血模型;18只大鼠建模成功后随机摸球法分为3组。空白组:损伤后6 h于局部注射500μL EGM-2;对照组:损伤后6 h于局部注射500μL含1×10~6个HUCB-late EPCs的EGM-2;实验组:损伤后6 h于局部注射500μL含1×10~6个CXCR4过表达HUCB-late EPCs的EGM-2。28天后,HE染色检测缺血后肢肌肉间、肌肉旁微小血管情况;免疫组织化学染色检测肌肉间、肌肉旁新生血管CD31阳性内皮细胞的表达情况,并计算CD31阳性新生微血管数量。结果 rAAV_6-CXCR4感染组HUCB-late EPCs中CXCR4蛋白的表达高于rAAV_6-GFP感染组和对照组。3组体外成血管能力差异无统计学意义(P0.05)。体内实验,与空白组、对照组比较,实验组新生微血管表达的CD31~+内皮细胞明显增多(P0.05)。结论 CXCR4过表达的HUCB-late EPCs可以促进SD大鼠后肢缺血组织新生微血管的形成。     

Retention of endothelial progenitor cells in bone marrow in a murine model of endogenous tissue plasminogen activator (tPA) deficiency in response to critical limb ischemia

Background

This study tested the hypothesis that tissue plasminogen activator (tPA) is crucial for regulating endothelial progenitor cell (EPC) mobilization from bone marrow to circulation in murine critical limb ischemia (CLI) by ligating the left femoral artery.

Methods

Wild-type (C57BL/6) (n = 40) mice were equally divided into group 1A (sham control), group 2A (CLI), group 3A [control-tPA (4.0 mg/kg)] and group 4A [CLI-tPA (intravenously at 3 h after CLI)]. Similarly, tPA knock-out (tPA^−/−) mice (n = 40) were equally divided into group 1B (sham control), group 2B (CLI), group 3B [control-tPA (4.0 mg/kg)], and group 4B (CLI-tPA).

Results

The circulating levels of EPCs (C-kit/CD31 +, Sca-1/KDR +, CXCR4/CD34 +) were lower in groups 1B and 2B than in groups 1A and 2A, respectively (all p < 0.01), and were reversed after tPA treatment (3B vs. 3A or 4B vs. 4A, p > 0.05) at 6 h and 18 h post-CLI. Levels of these biomarkers decreased again 14 days after CLI in tPA^−/− mice compared to those in wild-type between the respective groups (all p < 0.01). Laser Doppler flowmetry showed a higher ratio of ischemic-to-normal blood flow in 2A than in 2B and in 4A than in 4B by day 14 after CLI (all p < 0.05). Angiogenesis at protein (CXCR4, SDF-1α, VEGF) and cellular (CXCR4 +, SDF-1α +, and CD31 + cells) levels was highest in animals with CLI-tPA, significantly higher in mice with CLI only than in sham controls for both wild-type and tPA^−/− mice (p < 0.01).

Conclusion

tPA played an essential role in augmenting circulating EPCs, angiogenesis, and blood flow in the ischemic limb in a murine model.     

In vivo vasculogenic potential of human blood-derived endothelial progenitor cells

Vascularization of tissues is a major challenge of tissue engineering (TE). We hypothesize that blood-derived endothelial progenitor cells (EPCs) have the required proliferative and vasculogenic activity to create vascular networks in vivo. To test this, EPCs isolated from human umbilical cord blood or from adult peripheral blood, and human saphenous vein smooth muscle cells (HSVSMCs) as a source of perivascular cells, were combined in Matrigel and implanted subcutaneously into immunodeficient mice. Evaluation of implants at one week revealed an extensive network of human-specific lumenal structures containing erythrocytes, indicating formation of functional anastomoses with the host vasculature. Quantitative analyses showed the microvessel density was significantly superior to that generated by human dermal microvascular endothelial cells (HDMECs) but similar to that generated by human umbilical vein endothelial cells (HUVECs). We also found that as EPCs were expanded in culture, their morphology, growth kinetics, and proliferative responses toward angiogenic factors progressively resembled those of HDMECs, indicating a process of in vitro maturation. This maturation correlated with a decrease in the degree of vascularization in vivo, which could be compensated for by increasing the number of EPCs seeded into the implants. Our findings strongly support the use of human EPCs to form vascular networks in engineered organs and tissues.     

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。     

内皮祖细胞移植治疗糖尿病兔下肢缺血的影像学改变

目的探讨内皮祖细胞（EPC）移植治疗糖尿病兔模型下肢缺血的疗效。方法将日本大耳白兔随机分3组：糖尿病PBS对照组（A组）、糖尿病内皮祖细胞移植治疗组（B组）、正常血糖内皮祖细胞移植治疗组（C组）。骨髓来源的内皮祖细胞经体外扩增培养7d后,通过肌内注射进行细胞移植。结果EPC移植后14d,彩超示B组兔缺血侧／正常侧下肢胫前动脉收缩期峰值流速比值明显增加,动脉造影示该组缺血侧下肢动脉显影血管数多于对照组（P〈0．05）。结论EPC移植能有效治疗糖尿病兔模型下肢缺血。     

Enrichment of hematopoietic progenitor cells from human bone marrow on percoll density gradients

Summary Hematopoietic progenitor cells were fractionated on continuous and discontinuous Percoll density gradients. It could be shown, that Percoll provides a suitable gradient medium for analytical as well as preparative density gradient procedures without major methodological problems. Advantages compared to albumin and Ficoll-Metrizoate are discussed.