外周血中循环血管内皮细胞及血管内皮前体细胞的扩增与鉴定

目的证实外周血中循环血管内皮细胞(circulating endothelial cells,CECs)及血管内皮前体细胞(circulating endothelial precursors,CEPs)的存在,以及内皮前体细胞的增殖分化能力.方法应用流式细胞法检测57例肿瘤患者及15例正常人外周血中的CECs/CEPs.选择经造血干细胞动员的肿瘤患者,分离单个核细胞(mononuclear cells,MNCs),在VEGF、IGF及bFGF条件下培养血管内皮细胞,并应用透射电镜法对培养细胞进行鉴定.结果肿瘤患者外周血中CECs/CEPs含量分别为0.378±0.047%、0.059±0.013%,高于正常对照组.培养细胞透射电镜下可见Weibel-Palade小体,为血管内皮细胞的特异性标志.结论外周血中存在CECs/CEPs,在肿瘤患者体内存在血管内皮及其前体细胞的动员,循环血管内皮干/祖细胞可进一步分化成熟.     

肿瘤患者外周血循环血管内皮细胞数量与血清VEGF水平的关系

目的：分析实体瘤患者外周血中的循环血管内皮细胞（circulating endothelial cells, CECs）及血管内皮前体细胞(circulating endothelial precursors，CEPs)，并比较与正常人的差异。方法：流式细胞法检测57肿瘤患者及15例正常人外周血中的CECs和CEPs，ELISA法检测血清中血管生成相关因子VEGF和bFGF的表达水平。结果：肿瘤患者外周血CECs及CEPs分别为(0.378±0.047)%和(0.059±0.013)%，血清VEGF，bFGF分别为(295.58±59.56) ng/L和(28.73±7.40) ng/L，均高于正常对照组（P<0.05）。血管内皮及其前体细胞的数量与血清VEGF，bFGF水平相关。结论：实体瘤患者外周血中循环血管内皮及其前体细胞、血管内皮生长因子均高于正常人；VEGF和bFGF可能参与了血管内皮干/祖细胞的动员过程。     

血管内皮抑制素对Calu-6裸鼠移植瘤的生物学效应

目的 探讨血管内皮抑制素对Calu-6裸鼠移植瘤生长及新生血管形成的影响.方法 在荷瘤裸鼠皮下注射不同剂量的血管内皮抑制素,观察注射后肿瘤体积的变化;应用免疫组化SABC法检测肿瘤组织中血管内皮生长因子(VEGF)、生存素(survivin)和环氧化酶-2(COX-2)蛋白的表达以及微血管密度(MVD)的变化;流式细胞术检测循环血管内皮细胞(CECs)的含量;应用逆转录聚合酶链反应(RT-PCR)和实时定量PCR检测外周血中CD146和CD105 mRNA的表达.结果经血管内皮抑制素治疗后,荷瘤鼠肿瘤体积明显减小;肿瘤组织中VEGF、survivin和COX-2蛋白的表达以及MVD均下降,且各治疗组与阳性对照组间的差异均有统计学意义(均P＜0.05);外周血中CECs、CD146和CD105 mRNA的含量均明显下降;活化CECs的含量与肿瘤组织中survivin和VEGF的表达以及MVD的变化均呈正相关.结论 血管内皮抑制素可通过下调移植瘤中VEGF、survivin和COX.2蛋白的表达以及减少MVD抑制肿瘤生长;活化CECs将可能作为理想的预测抗血管形成治疗预后的标记物应用于临床.     

重组人血管内皮抑制素联合NP化疗对Ⅳ期非小细胞肺癌患者无进展生存期的影响

目的 探讨重组人血管内皮抑制素注射液联合NP方案(长春瑞滨+顺铂)治疗Ⅳ期非小细胞肺癌(NSCLC)患者的效果。方法 选取Ⅳ期NSCLC患者80例作为研究对象,依据随机数字表法分为NP组、联合组,各40例。NP组采用NP方案治疗,联合组在NP组基础上采用重组人血管内皮抑制素注射液治疗。比较两组疗效、治疗前、治疗3个疗程后肿瘤标志物[血清血管内皮生长因子(VEGF)、癌胚抗原(CEA)、糖类抗原125(CA125)]水平、循环内皮细胞[外周血活性循环内皮细胞(a CECs)、循环内皮细胞(CECs)]水平、中位无进展生存期、不良反应(恶心呕吐、肝肾功能损伤、粒细胞减少、贫血等)发生率。结果 联合组治疗总有效率(47.50%)(19/40)高于NP组(25.00%)(10/40),中位无进展生存期长于NP组(P<0.05);治疗3个疗程后,两组血清VEGF、CEA、CA125水平均下降,联合组低于NP组(P<0.05);治疗3个疗程后,联合组外周血a CECs、CECs水平下降(P<0.05),NP组无明显变化(P>0.05),联合组低于NP组(P<0.05)...     

肺癌患者外周血循环内皮细胞和VEGF检测临床意义的探讨

目的:探讨肺癌患者外周血循环内皮细胞(CECs)包括活性循环内皮细胞(aCECs)与VEGF之间的关系及其在肺癌中的临床意义。方法:用流式细胞仪对70例肺癌患者(NSCLC64例)化疗前后及30例健康人外周血CECs及aCECs进行检测,同时采用ELISA方法检测肺癌患者化疗前后VEGF水平。结果:NSCLC患者外周血CECs及aCECs的数量明显高于健康人群(P=0.000)。不同病理类型肺癌之间CECs及aCECs的数量差异无统计学意义(NSCLC与SCLC,P=0.878、0.936;鳞癌与腺癌,P=0.786、0.658)。CECs及aCECs的数量与VEGF之间无明显相关性(P=0.697、0.631)。治疗后不同疗效间肺癌患者VEGF水平比较,PD均高于CR+PR及SD(P=0.009、0.031)。NSCLC及SCLC化疗后aCECs的数量高于化疗前(P=0.031、0.029);SD及PD患者CECs、aCECs的数量高于化疗前(P=0.024、0.014;P=0.036、0.028);对治疗后不同疗效肺癌患者CECs及aCECs水平比较,aCECs数量PD、SD均高于CR+PR(P=0.022、0.009)。结论:VEGF联合CECs及aCECs检测可以作为很好的预测性指标评估肺癌的血管生成状况及判断预后。     

肿瘤相关循环内皮细胞的分类标志物及其功能研究进展

经典肿瘤新生血管的形成主要包括血管生成(angiogenesis)和血管发生(vasculogenesis)、均依赖循环内皮细胞(circulating endothelial cells,CECs)的增殖、迁移、黏附而形成管腔.CECs不仅与血管新生和肿瘤生长、播散有关,还可能作为抗血管生成药物的靶点和反映疗效的标志物.循环内皮细胞膜上可表达多种表面分子,在从幼稚到成熟及活化过程要经历不同的阶段,而不同阶段细胞表达的表面分子有所不同.各表面分子在肿瘤血管新生过程中发挥重要作用.监测可靠的分子标志物量化CECs尤其是活化血管内皮细胞(activated circulating vascular endothelial cells,aCECs),来判定肿瘤新生血管和血管靶向治疗的疗效具有重要意义且已成为临床上的迫切需要.本文就肿瘤相关循环内皮细胞的分类、标志物及其功能做一综述.     

循环内皮细胞与肿瘤血管生成

循环内皮细胞（circulating endothelial cells，CECs）是指外周血中测得的血管内皮细胞（vascular endothelial cells，VEC）。20世纪70年代，Bou—vier和Hladovec在动物试验中发现了这种细胞，并提出了CECs的概念。CECs在正常人外周血中数量极少，而在动脉粥样硬化、糖尿病、红斑狼疮等疾病中明显增加。多项研究表明，CECs数量的增加与内皮细胞受损明显相关，因此被认为是判断血管内皮细胞损伤情况的最特异而直接的指标。     

循环内皮祖细胞及其与肿瘤关系的研究进展

内皮祖细胞(endothelial progenitor cell, EPC)是存在于骨髓和循环外周血的祖细胞亚群，具有在体内外分化为成熟内皮细胞的能力，参与血管发生和血管稳态。恶性肿瘤细胞产生多种细胞因子,使骨髓中的EPC动员至外周血,并募集到肿瘤组织的血管床,参与肿瘤血管生成、肿瘤生长和转移。外周血EPC水平与肿瘤的体积、抗新生血管治疗的反应性和预后等密切相关，因此EPC可作为肿瘤血管新生的生物标记物。EPC还可作为靶向肿瘤的细胞载体负载自杀基因、毒素或药物，为抗肿瘤治疗提供了新的途径。     

胃癌患者血清VEGF水平与T淋巴细胞嗜银蛋白含量的相关性及临床意义

目的检测胃癌患者血清血管内皮生长因子(VEGF)水平和外周血T淋巴细胞嗜银蛋白(Ag-NORs)含量,探讨二者间的相关性及临床意义。方法采用酶联免疫双抗夹心法(ELISA)定量检测74例胃癌患者和32例健康人血清VEGF水平,同时检测外周血T淋巴细胞嗜银蛋白含量并进行相关分析。结果胃癌患者血清VEGF水平(383.33±121.67)pg/ml明显高于健康人(142.86±71.29)pg/ml(P<0.01),其水平与肿瘤大小、浸润深度、分化程度、有无转移、TNM分期有关(P<0.05或0.01);而胃癌患者外周血T淋巴细胞Ag-NORs含量(4.30±1.21)%明显低于健康人(7.65±1.38)%(P<0.01),其含量与胃癌分化程度、有无转移、TNM分期有关(P<0.05或0.01)。胃癌患者血清VEGF水平与外周血T淋巴细胞嗜银蛋白含量呈显著负相关(r=-0.659,P<0.01)。结论胃癌患者血清VEGF水平和外周血T淋巴细胞Ag-NORs含量是评估胃癌恶性生物学行为的重要指标。二者密切相关,对评价机体肿瘤免疫功能具有重要的临床参考价值。     

非小细胞肺癌患者血清血管内皮生长因子水平及临床意义

血管内皮生长因子(vascular endothelium growth factor, VEGF)是一种具有内皮细胞特异性的有丝分裂原,在肿瘤血管生成中有重要作用[1].为评价非小细胞肺癌(non-small cell lung cancer,NSCLC)患者血清VEGF水平变化的临床意义,我们检测了患者血清VEGF的水平,探讨其与肺癌发展和临床分期之间的关系.     

Increased levels of viable circulating endothelial cells are an indicator of progressive disease in cancer patients.

BACKGROUND: There is accumulating evidence from preclinical studies that circulating endothelial cells (CECs) play an important role in neovascularization and tumor growth. The role of CECs in human cancer progression is sparsely investigated. We therefore analyzed CECs in peripheral blood of cancer patients. In addition, we correlated CEC levels in these patients with plasma levels of cytokines that are known to mobilize CECs in experimental models. PATIENTS AND METHODS: Viable CECs were isolated, quantified and cultured from cancer patients' whole blood by using magnetic beads coupled to an antibody directed against CD146, a pan-endothelial marker. Viable cells were visualized by calceinAM staining. Positive staining for specific endothelial cell markers [i.e. von Willebrand factor, CD31, vascular endothelial cell growth factor (VEGF) receptor-2] was used to confirm the endothelial phenotype. RESULTS: Cancer patients with progressive disease (95 patients) had on average 3.6-fold more CECs than healthy subjects (46 patients, P <0.001). Patients (17) with stable disease had CEC numbers equal to that circulating in healthy subjects (P = 0.69). A subset of in vitro cultured CECs incorporated into endothelial layers and formed colonies. Plasma levels of cytokines that are thought to mobilize CECs from the bone marrow [VEGF, placental growth factor, stromal cell derived factor 1alpha and stem cell factor (71 patients)] did not correlate with CEC amounts. The levels of viable CECs in cancer patients were modified by granulocyte colony-stimulating factor treatment and chemotherapy. CONCLUSION: In progressive cancer patients, the amount of CECs is increased. These CECs are viable and may contribute to vessel formation. The number of CECs is influenced by anticancer treatment.     

Differential effects of vascular endothelial growth factor receptor-2 inhibitor ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: implications for use as a surrogate marker of antiangiogenic activity.

PURPOSE: Circulating endothelial cells (CEC) comprise at least two distinct populations: bone marrow-derived circulating endothelial progenitors (CEP) and mature CECs derived from existing vasculature. We hypothesized that antiangiogenic agents may have differential effects on CEPs and mature CECs and that these changes may serve as a marker of biological activity. EXPERIMENTAL DESIGN: The effect of angiogenesis inhibitors on CECs was evaluated by flow cytometry after vascular endothelial growth factor (VEGF)-induced mobilization and in mice bearing Lewis lung carcinoma (LLC). Tumor angiogenesis was evaluated in parallel by immunohistochemistry. RESULTS: In nontumor-bearing mice, VEGF administration increased both mature CECs and CEPs. This increase was inhibited by the VEGF receptor 2 inhibitor ZD6474 as well as the VEGF inhibitor-soluble Flt-1. ZD6474 had no significant effect on CECs in the absence of exogenous VEGF stimulation. In contrast, LLC-bearing mice had an increase in mature CECs but not CEPs after 3 days of treatment with ZD6474. The increase in mature CECs was dose-dependent, accompanied by a decrease in tumor microvessel density, and preceded reduction in tumor volume. Treatment of LLC-bearing mice with the vascular targeting agent ZD6126 also increased mature CECs. CONCLUSIONS: VEGF inhibitors can have differential effects on mature CECs and CEPs, and agents inhibiting tumor angiogenesis may cause a concomitant increase in mature CECs. This increase occurs in tumor-bearing but not in nontumor-bearing mice, suggesting that tumor endothelium is a potential source of mature CECs. Therefore, assessing both mature CECs and CEPs may provide insights into the mechanism of antiangiogenic agents and serve as an early surrogate marker of biological activity.     

Endothelial precursors and mature endothelial cells are increased in the peripheral blood of myelodysplastic syndromes

Increased angiogenesis has been demonstrated to be a significant prognostic factor in many solid tumors. In the oncohematological setting, it has been associated with myelodysplastic syndromes (MDS), chronic myeloid leukemia, acute lymphoid, and myeloid leukemias. Recently, increased circulating endothelial cells (CECs) have been associated with breast cancer and non-Hodgkin lymphoma (NHL). Based on these premises we analysed total and activated CECs, and endothelial precursors (CEPs) in 50 MDS patients and 20 healthy donors. CECs and CEPs were quantified by flow cytometry. CEC levels were compared with bone marrow (BM) microvessel density (MVD). In addition, some angiogenic factors, namely vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble VEGF-Receptor2 (VEGFR2), were tested in the sera from 25 MDS patients. Total, activated CECs and CEPs were significantly increased in MDS when compared to control group (p<0.0001); whereas in the MDS cases no association was found with French--American--British (FAB), International Prognostic Scoring System (IPSS) subtypes or survival. Patients with higher CECs also showed higher MVD. Among the cytokines analysed, sVEGFR2 was significantly higher in the lower IPSS risk classes, while the levels of bFGF directly correlated with total and activated CECs. Taken together these data strengthen the hypothesis of a possible role of angiogenesis in MDS pathogenesis.     

Increased mobilisation of circulating endothelial progenitors in von Hippel-Lindau disease and renal cell carcinoma

Background:

Circulating endothelial cells (CECs) are a candidate biomarker for monitoring angiogenesis in cancer. Circulating endothelial cell subsets are mobilised by angiogenic mediators. Because of the highly angiogenic phenotype of renal cell carcinoma (RCC), we sought to assess the potential of CECs as a marker of RCC in patients with von Hippel-Lindau (VHL) disease and those with sporadic RCC.

Methods:

We performed multicolour flow cytometry to enumerate CECs in patients with RCC, patients with VHL disease with and without RCC, and normal subjects. Two subsets of CECs were evaluated: mature CECs (mCECs) and circulating endothelial progenitors (CEPs).

Results:

In patients with VHL disease and RCC and those with sporadic RCC (N=10), CEPs and the CEP:mCEC ratio were higher than in normal subjects (N=17) (median CEPs: 0.97 vs 0.19 cells μl⁻¹, respectively, P<0.01; median CEP:mCEC: 0.92 vs 0.58, respectively, P=0.04). However, in patients with VHL without RCC, CECs were not increased. In paired pre- and post-nephrectomy RCC patient samples (N=20), CEPs decreased after surgery (median difference 0.02 cells μl⁻¹, −0.06 to 1.2; P=0.05).

Conclusion:

Circulating endothelial progenitors were elevated in RCC, but not in patients with VHL without RCC. Circulating endothelial progenitor enumeration merits further investigation as a monitoring strategy for patients with VHL.     

Circulating endothelial progenitors and CXCR4-positive circulating endothelial cells are predictive markers for bevacizumab

BACKGROUND:

Bevacizumab plus chemotherapy is a standard option in the treatment of metastatic colorectal cancer (mCRC). The aim of this study was to investigate the potential of circulating endothelial cell progenitors (CEPs) and phenotypical circulating endothelial cells (CECs) as surrogate markers of clinical outcome in mCRC patients to identify responders to bevacizumab in combination with chemotherapy.

METHODS:

A total of 69 patients with measurable mCRC were enrolled in this prospective study. Whole blood samples were analyzed before initiation of treatment and on days 4 and 14. Phenotypical CECs and CEPs were then isolated and enumerated by using flow cytometry.

RESULTS:

CEP levels of less than 0.04% on day 4 were significantly associated with longer progression‐free survival (PFS) and overall survival (OS) (P < .001, P = .002, respectively) as compared with levels of 0.04% or more. In addition, CXCR4‐positive CEC levels of less than 20% at baseline were significantly associated with longer PFS and OS as compared other indicators investigated (P < .001, P = .002, respectively).

CONCLUSIONS:

Levels of CEPs on day 4 and proportion of CXCR4‐positive CECs at baseline were correlated with the prognosis of bevacizumab combination chemotherapy, suggesting that these surrogate markers may play a core role in the selection of candidates for bevacizumab treatment. Cancer 2011;. © 2011 American Cancer Society.     

Evaluation of circulating endothelial cells, VEGF and VEGFR2 serum levels in patients with chronic myeloproliferative diseases

Authors evaluated some markers of angiogenetic activity in patients with chronic myeloproliferative diseases (CMDs). In this study by using a cytofluorimetric analysis we evaluated circulating endothelial progenitor cells (EPCs) in patients with chronic myeloproliferative disease. Moreover, in the same group of subjects, we evaluated serum levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR2). In our patients, we have found an increase in the number of endothelial progenitor cells in primary myelofibrosis (PMF) and polycythaemia vera (PV) patients, while an increase of circulating endothelial cells (CECs) was found in all patients with CMD. Moreover, we found higher serum levels of VEGF with respect to control subjects in every group of patients with CMD, and a not significant reduction of VEGFR2 levels in essential thrombocythaemia (ET) patients. A correlation was also found in PV patients between VEGF levels and erythrocyte number and in PMF subjects with the count of white cells. Our data suggest that some markers of angiogenesis are activated in CMD patients and angiogenesis may have a role in the pathophysiology of chronic myeloproliferative disorders.     

Genetic heterogeneity of the vasculogenic phenotype parallels angiogenesis; Implications for cellular surrogate marker analysis of antiangiogenesis

Development of antiangiogenic therapies would be significantly facilitated by quantitative surrogate pharmacodynamic markers. Circulating peripheral blood endothelial cells (CECs) and/or their putative progenitor subset (CEPs) have been proposed but not yet fully validated for this purpose. Herein, we provide such validation by showing a striking correlation between highly genetically heterogeneous bFGF- or VEGF-induced angiogenesis and intrinsic CEC or CEP levels measured by flow cytometry, among eight different inbred mouse strains. Moreover, studies using genetically altered mice showed that levels of these cells are affected by regulators of angiogenesis, including VEGF, Tie-2, and thrombospondin-1. Finally, treatment with a targeted VEGFR-2 antibody caused a dose-dependent reduction in viable CEPs that precisely paralleled its previously and empirically determined antitumor activity.