CD146-based immunomagnetic enrichment followed by multiparameter flow cytometry: a new approach to counting circulating endothelial cells

Summary. Background: Circulating endothelial cells (CECs) have emerged as non‐invasive biomarkers of vascular dysfunction. The most widely used method for their detection is CD146‐based immunomagnetic separation (IMS). Although this approach has provided consensus values in both normal and pathologic situations, it remains tedious and requires a trained operator. Objectives: Our objective was to evaluate a new hybrid assay for CEC measurement using a combination of pre‐enrichment of CD146+ circulating cells and multiparametric flow cytometry measurement (FCM). Patients and methods: CECs were determined in peripheral blood from 20 healthy volunteers, 12 patients undergoing coronary angioplasty, and 30 renal transplant recipients, and blood spiked with cultured endothelial cells. CD146+ cells were isolated using CD146‐coated magnetic nanoparticles and labeled using CD45–fluorescein isothiocyanate and CD146–PE or isotype control antibody and propidium iodide before FCM. The same samples were also processed using CD146‐based immunomagnetic separation as the reference method. Results: The hybrid assay detected CECs, identified as CD45^dim/CD146^bright/propidium iodide⁺, with high size‐related scatter characteristics, and clearly discriminated these from CD45^bright/CD146^dim activated T lymphocytes. The method demonstrated both high recovery efficiency and good reproducibility. Both IMS and the hybrid assay similarly identified increased CEC levels in patients undergoing coronary angioplasty and renal transplantation, when compared to healthy controls. In patients, CEC values from these two methods were of the same order of magnitude and highly correlated. Bland–Altman analysis revealed poor statistical agreement between methods, flerrofluid–FCM providing higher values than IMS. Conclusion: This new hybrid FCM assay constitutes an accurate alternative to visual counting of CECs following CD146‐based IMS.     

Quantitative detection of circulating endothelial cells in vasculitis: comparison of flow cytometry and immunomagnetic bead extraction

Summary.  Background:  Circulating endothelial cells (CECs) are biomarkers for endothelial cell (EC) injury and are quantified using immunomagnetic bead extraction (IBE), or flow cytometry (FC). Reports suggest that there is good agreement between these methods for CEC quantification. Objectives:  We examined levels of agreement between these techniques in children with systemic vasculitis. Methods:  We added HUVEC or human pulmonary artery EC to whole blood to optimize FC gating strategies for EC. EC-optimized FC was then compared with IBE for CEC enumeration in 25 children with vasculitis and 20 healthy controls. Results:  Using Bland–Altman analysis, agreement between IBE and EC-optimized FC was poor in children with vasculitis ( n  = 25) and healthy controls ( n  = 20): IBE consistently detected higher values than the EC-optimized FC method: the mean difference between the two techniques was 60 CECs mL⁻¹, 95% CI ±374 CECs mL⁻¹ (paired analyses of 45 individuals). Agreement was poorest for vasculitis patients: mean difference (IBE – EC-optimized FC) 120 CECs mL⁻¹, 95% CI ±460 CECs mL⁻¹ ( P  = 0.018). We identified three reasons for this discrepancy: (i) sub-optimal FC gating parameters previously used for detecting CECs; (ii) inherent lack of sensitivity of FC compared with IBE for CEC rare event detection; and (iii) use of lysis buffers required for FC causing CEC lysis. Conclusions:  There was poor agreement between EC-optimized FC and IBE for the quantification of CECs from children with active vasculitis and controls. We emphasize that in this clinical setting the two techniques are not directly comparable when comparing results obtained using these different methodologies.     

A new approach for rapid and reliable enumeration of circulating endothelial cells in patients

Background:  Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4–1300 per mL). Objectives:  Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi‐parameter flow cytometric (FCM) method without immunological pre‐enrichment. Methods:  CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein‐wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell μL^?1. Results:  The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch^®). CEC levels ranged from 4 to 79 CEC mL^?1 in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001). Conclusions:  This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.     

低氧血症新生儿循环内皮细胞数量的变化及其临床意义

目的：探讨低氧血症新生儿外周血循环内皮细胞（CECs）水平变化及其临床意义。方法采用 Hladovec 计数法测定40例不同程度缺氧的低氧血症患儿（低氧血症组）及20例血氧分压正常的健康新生儿（对照组）外周血 CECs 数量。结果低氧血症组 CECs 为（0．910±0．422）×106／L 与对照组比较,高于对照组的（0．180±0．100）×106／L,差异有统计学意义（t＝7．539, P ＜0．05）;中度低氧血症组 CECs 为（1．140±0．135）×106／L,高于轻度低氧血症组的（0．540±0．127）×106／L,差异有统计学意义（t＝13．43,P ＜0．05）;重度低氧血症组 CECs 为（1．660±0．114）×106／L,高于中度低氧血症组的（1．140±0．135）×106／L,差异有统计学意义（t＝7．698,P ＜0．05）。结论外周血 CECs 数量可反映低氧血症患儿缺氧严重程度,可作为低氧血症的早期诊断指标,为临床早期诊断、病情判断及疗效评估提供新的判断依据。     

循环肿瘤细胞的分离与鉴定研究进展

循环肿瘤细胞(CTC)是由自发或诊疗操作中从实体瘤 、转移灶进入外周血循环系统的肿瘤细胞,是"液体活检"的一项重要指标,可以为肿瘤的早期诊断 、转移复发的评估提供有力帮助.但由于体液中CTC的数量极少,检测CTC首先需要将细胞高效分离出来,并对细胞纯度 、表型进行鉴定.目前已经建立了较多分离 、鉴定CTC的方法,本文就...     

Circulating microparticles and endothelial progenitor cells in atherosclerosis: pharmacological effects of irbesartan

Summary. Aims: This study aimed to (i) employ our newly designed model, the hypertensive–hypercholesterolemic hamster (HH), in order to find out whether a correlation exists between circulating microparticles (MPs), endothelial progenitor cells (EPCs) and their contribution to vascular dysfunction and (ii) to assess the effect of irbesartan treatment on HH animals (HHI).Methods and Results: The results showed that compared with the control (C) group, HH displayed: (i) a significant increase in plasma cholesterol and triglyceride concentration, and an augmentation of systolic and diastolic arterial blood pressure, and of heart rate; (ii) a marked elevation of MPs and a significant decrease in EPCs; (iii) structural modifications of the arterial wall correlated with altered protein expression of MMP2, MMP9, MMP12, TIMP1, TIMP2 and collagen type I and III; (iv) a considerably altered reactivity of the arterial wall closely correlated with MPs and EPC adherence; and (v) an inflammatory process characterized by augmented expression of P‐Selectin, E‐Selectin, von Willebrand factor, tissue factor, IL‐6, MCP‐1 and RANTES. Additionally, the experiments showed the potential of irbesartan to correct all altered parameters in HH and to mobilize EPCs by NO, chemokines and adhesion molecule‐dependent mechanisms.Conclusions: Hypertension associated with hypercholesterolemia is accompanied by structural modifications and expression of pro‐inflammatory molecules by the vessel wall, the alteration of vascular tone, enhanced release of MPs and reduced EPCs; the ratio between the latter two may be considered as a marker of vascular dysfunction. Irbesartan, which exhibits a pharmacological control on the levels of MPs and EPCs, has the potential to restore homeostasis of the arterial wall.     

The relationship of circulating endothelial cells to plasma indices of endothelial damage/dysfunction and apoptosis in acute coronary syndromes: implications for prognosis

Summary. Background: While coronary artery disease has been linked to both endothelial damage and cellular apoptosis, their inter‐relationships and impact on cardiovascular (CV) outcomes has been barely explored. Aims: First, we investigated the inter‐relationships between circulating endothelial cells (CECs, and index of endothelial damage) and circulating plasma markers of endothelial damage/dysfunction [von Willebrand factor (VWF), soluble E selectin (sEsel)] and apoptosis [soluble Fas (sFas), Fas ligand (sFasL) and their ratio, sFas/sFasL] in patients presenting with acute coronary syndrome (ACS). Second, we assessed their prognostic values for major adverse CV events (MACE) in ACS. Methods: We studied 211 patients with ACS, who were compared with 60 healthy controls (HC) and 45 ‘disease controls’ (patients with stable coronary artery disease, CAD). Simultaneous blood samples for CECs (immunobead method), VWF, sESel, sFas and sFasL (ELISA) were taken within 24 h of presentation of ACS and at 48 h post admission. Results: CEC, sEsel and VWF levels were significantly higher among the ACS groups compared with the CAD and HC (P < 0.05) groups. sFas was higher (P = 0.016) and sFasL lower (P = 0.021) in ACS compared with controls (HC and CAD). There was a significant increase in sFas/sFasL ratio with increasing disease severity (P = 0.0004). There were significant correlations between CECs and both VWF and sEsel (both P < 0.01) but no correlations between CECs and either sFas or sFas ligand. On univariate survival analysis, CECs were associated with an increased risk of both MACE [hazard ratio (HR) 2.4 (95% CI 1.2–4.1); P = 0.009] and cardiovascular death [HR 2.95 (95% CI 1.01–8.81); P = 0.047]. On multivariate Cox regression analysis, only VWF (and not CECs) remained as an independent predictor of MACE [HR 1.02 (95% CI 1.005–1.040); P = 0.009]. Conclusion: CECs were associated with abnormal plasma indices of endothelial damage/dysfunction and not apoptosis, despite abnormalities of all these markers being associated with ACS. VWF remained as an independent predictor of MACE, on multivariate analysis.     

Ⅰ型糖尿病患儿循环内皮细胞计数检测与尿白蛋白相关性研究

目的探讨Ⅰ型糖尿病患儿血管内皮损害标志物循环内皮细胞计数的变化,以及与尿白蛋白的相关性。方法将62例Ⅰ型糖尿病患儿根据尿白蛋白排泄率分为正常白蛋白尿组（A组）35例和微量白蛋白尿组（B组）27例,抽取同期健康体检儿童60例作为对照组（C组）,分别检测其循环内皮细胞计数和尿微量蛋白、糖化血红蛋白的变化,并进行相关性分析。结果Ⅰ型糖尿病患儿循环内皮细胞计数及糖化血红蛋白均显著高于正常对照组（P〈0．01）;微量白蛋白尿组糖尿病患儿均显著高于正常白蛋白尿组患儿（P〈0．05）;Ⅰ型糖尿病患者循环内皮细胞计数与糖化血红蛋白呈显著正相关（R=0．310,P〈0．05）。结论Ⅰ型糖尿病患儿在出现微量白蛋白尿前已存在血管内皮功能异常,循环内皮细胞计数的检测可作为早期筛查糖尿病肾病的可靠指标。     

Improving cardiovascular outcomes in rheumatic diseases: Therapeutic potential of circulating endothelial progenitor cells

Patients with Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE) have a significantly increased risk of cardiovascular disease (CVD). The reason for this is unclear but may be due, at least in part, to the failure of endothelial repair mechanisms. Over the last 15 years there has been much interest in the mechanisms of endothelial renewal and its potential as a therapy for CVD. In the circulation there are two distinct populations of cells; myeloid angiogenic cells (MACs) which augment repair by the paracrine secretion of angiogenic factors, and outgrowth endothelial cells (OECs) which are true endothelial progenitor cells (EPCs) and promote vasculogenesis by differentiating into mature endothelium. There are marked abnormalities in the number and function of these cells in patients with RA and SLE. Inflammatory cytokines including interferon-alpha (IFNα) and tumour-necrosis factor alpha (TNFα) both impair MAC and OEC function ex vivo and may therefore contribute to the CVD risk in these patients. Whilst administration of mononuclear cells, MACs and other progenitors has improved cardiovascular outcomes in the acute setting, this is not a viable option in chronic disease. The pharmacological manipulation of MAC/OEC function in vivo however has the potential to significantly improve endothelial repair and thus reduce CVD in this high risk population.     

循环肿瘤细胞的磁分离法建立及在肺癌EGFR-TKI治疗中的应用

目的探索磁分离循环肿瘤细胞(circulating tumor cells,CTCs)联合竞争性等位基因特异性Taq ManPCR(Cast PCR)检测表皮生长因子受体(EGFR)基因突变的可行性。方法收集12例晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)患者血液样本各7.5 m L,两步法磁分离CTCs,免疫荧光染色检测上皮细胞黏附分子(Ep CAM)和细胞角蛋白(Cytokeratin)后,流式细胞术检测CTCs数量及纯度;Cast PCR检测CTCs及循环DNA中EGFR基因del EGFR19、T790M和L858R突变;用EGFR基因突变检测试剂盒(ADx-ARMS法)检测癌组织的上述位点突变。结果高表达与不表达Ep CAM的CTCs分别在6例和12例NSCLC患者中被检测出;del EGFR19、T790M和L858R突变例数分别在2例、8例和5例NSCLC患者的CTCs中被检测出,其总检出率为83.3%(10/12);2例患者的循环DNA中检出L858R突变;ADx-ARMS法检测12例NSCLC患者癌组织中上述3种突变分别是2例、5例和3例,总检出率为75.0%(9/12)。CTCs与癌组织的突变具有一致性(Kappa=0.75,P=0.007),循环DNA与癌组织的突变一致性无统计意义(Kappa=0.06,P=0.546)。结论磁分离CTCs联合Cast PCR能有效检测EGFR基因突变,值得在NSCLC患者的EGFR酪氨酸激酶抑制剂(EGFR-TKI)治疗中推广应用。     

Levels of circulating endothelial progenitor cells are related to uremic toxins and vascular injury in hemodialysis patients

Summary.  Background : Patients suffering from chronic kidney diseases (CKD) exhibit cardiovascular diseases and profound endothelial dysfunction. CKD patients have reduced numbers of endothelial progenitor cells, but little is known about the factors influencing these numbers. Objectives : Among these factors, we hypothesized that uremic toxins and vascular injury affect endothelial progenitor cells. Patients/methods : Thirty-eight hemodialysis patients were investigated and compared with 21 healthy controls. CD34+CD133+ immature progenitors, CD34+KDR+ endothelial progenitors cells (EPC) and myeloid EPC (mEPC) were counted in peripheral blood. Levels of uremic toxins β₂-microglobulin, indole-3 acetic acid, indoxylsulfate, p-cresylsulfate and homocysteine were measured. Vascular injury was assessed in hemodialysis (HD) patients by measuring aortic pulse wave velocity and plasma levels of endothelial microparticles. In vitro experiments were performed to study the effect of uremic toxins on apoptosis of progenitor cells. Results and conclusions : CD34+CD133+ immature progenitor cell number was negatively correlated with the levels of uremic toxins β₂-microglobulin and indole-3 acetic acid. In vitro , indole-3 acetic acid induced apoptosis of CD133+ cells. These data indicate uremic toxins have a deleterious role on progenitor cells, early in the differentiation process. Moreover, mEPC number was positively correlated with markers of vascular injury–pulse wave velocity and endothelial microparticle levels. This suggests that vascular lesions could stimulate progenitor cell mobilization, even in a context of reduced EPC induced by CKD. In conclusion, uremic toxins and vascular injury appear to affect endothelial progenitor cell biology in CKD.     

体外诱导脂肪干细胞向内皮细胞及成血管的分化

背景：脂肪干细胞作为组织工程中种子细胞的选择,可以诱导为内皮细胞从而有效解决材料血管化困难的难题。目的：体外观察脂肪干细胞经诱导向内皮细胞转分化的可能性、以及在立体培养基上的血管形成情况。方法：切取人皮下脂肪,用酶消化法分离和培养脂肪干细胞,将传至第3代或第4代的脂肪干细胞用内皮细胞诱导液、同时在Matrigel三维培养基内诱导培养,观察细胞生长情况及变化。对脂肪干细胞和诱导细胞行免疫组织化学检查CD31的表达。结果与结论：免疫组织化学检测脂肪干细胞的CD31表达阴性,诱导细胞CD31可见阳性表达。在三维立体培养基内诱导的细胞24h逐渐迁徙成团,伸出伪足,诱导1周细胞形成网格样交叉,2周形成较长血管,后血管增粗,并出现分叉,CD31阳性表达。因此,脂肪干细胞体外可以被诱导向内皮细胞表型转分化,并形成血管,提示脂肪干细胞可以作为促进组织工程移植物血管化的种子细胞的理想选择。     

大鼠骨髓源性内皮祖细胞的分离培养与鉴定

背景：内皮祖细胞因其分离与培养的方法各不相同,在实验中难以重复。目的：探讨大量获取骨髓源性内皮祖细胞分离与培养的方法。方法：通过密度梯度离心法从4周龄SD大鼠骨髓中分离单个核细胞,使用EGM-2MV培养基进行诱导培养,采用形态学特征观察、摄取Dil-Ac-LDL与结合FITC—UEA-1实验、免疫荧光化学鉴定其表面抗原CD133与VEGFR2等方法对其进行鉴定,并通过管腔形成实验观察形成管腔的能力。结果与结论：①形态学观察：分离的骨髓单个核细胞经诱导培养后,在生长的早期（8d左右）、晚期（15d左右）其细胞形态有一定差异,早期以纺锤形、三角形、圆形细胞多见,晚期以圆形、短梭形细胞多见。②摄取Dil-Ac-LDL与结合FITC-UEA-1实验：显示8,21d的细胞均为阳性。⑧免疫荧光化学染色：8d的细胞表达CD133、VEGFR2。④管腔形成实验：在Matrigel基质上15h左右能够生成血管样结构。结果表明：利用密度梯度离心法分离大鼠骨髓单个核细胞后以EGM-2MV进行诱导培养,经过鉴定证明获得的细胞符合内皮祖细胞的特征。这种方法能够简单、快速、可靠、大量地获取内皮祖细胞。     

The effects of exercise stress testing on soluble E‐selectin,von Willebrand factor,and circulating endothelial cells as indices of endothelial damage/dysfunction

Background. The quantification of circulating endothelial cells (CECs) in whole blood is an increasingly recognized index of endothelial damage/dysfunction. Abnormal CECs have been linked to the severity of coronary artery disease (CAD).

Objective. We assessed the relationship of CECs to other markers of endothelial dysfunction (von Willebrand factor (vWF) and soluble E‐selectin (sEsel)) during exercise stress testing (EST) in a cohort of patients with suspected CAD.

Methods. We studied a cohort of patients referred to our chest pain clinic with a history of exertional chest pain. Treadmill EST was performed, using a full Bruce exercise protocol. Blood for CECs (immunobead method), vWF and sEsel (both ELISA) were collected immediately before (pre‐exercise), immediately following exercise, and at 30 minutes post‐EST.

Results. We studied 31 patients (84% male; mean (SD) age 58.4 (9.8) years). Of the entire cohort, 14 patients (45.2%) had a positive EST. Exercise led to significant increases in levels of CECs, sEsel, vWF, white blood cells (WBC), heart rate, mean and systolic blood pressure compared with base‐line (all P<0.05). There was a significant correlation between the change (Δ (immediate post–pre‐exercise)) in CECs and ΔvWF (r = 0.45; 95% CI 0.11–0.69: P = 0.01) and ΔsEsel (r = 0.41; 0.05–0.7: P = 0.02), as well as between ΔvWF and ΔsEsel (r = 0.55; 0.25–0.76: P = 0.001). Neither absolute nor ΔCEC counts were predictive of exercise work‐load/functional capacity, nor the presence of positive EST results.

Conclusion. EST led to a significant increase in endothelial markers (CECs, vWF, and sEsel) compared with base‐line levels. The rise in CECs correlated with the increases in other endothelial markers, but was not related to the either exercise work‐load/capacity or to the presence of a positive EST.     

小鼠肝星状细胞的分离纯化新方法的建立与应用

目的 介绍一种简便、经济、高产的小鼠肝星状细胞(HSC)分离方法,为肝纤维化的研究提供细胞模型.方法 参照国内外方法并加以改良,采用酶灌注预消化及随后的震荡充分消化,合并Medimachine系统制成单细胞悬液.用人淋巴细胞分离液直接铺梯度,采用单层梯度离心法一步分离HSC.结果 两只小鼠HSC得率可达1×106个,台盼蓝染色显示细胞活率达92%.初分离的HSC在328 nm激发光下自发蓝绿色荧光,油红染色及结蛋白免疫细胞化学染色鉴定纯度达90%.结论 建立了一种实用的小鼠HSC分离方法,可用于肝纤维化和原代HSC的生物学行为研究.该方法简便、实用、高产,不需特殊设备,便于推广应用.     

Proliferative glomerulonephritis in rats: evidence that mononuclear phagocytes infiltrating the glomeruli stimulate the proliferation of endothelial and mesangial cells

Abstract. A proliferative, non-crescentic, glomerulonephritis (GN) was induced in rats preimmunized with rabbit IgG by injecting a sub-nephrotoxic dose of rabbit anti-GBM IgG. Control rats either received anti-GBM IgG only, or were totally irradiated (800 rads, kidneys protected) 2 days before the second injection. All the GN rats developed a severe proteinuria within 2–4 days after the injection of anti-GBM IgG, contrarily to the control rats. At the same time, many mononuclear cells, of predominantly extra-renal origin, infiltrated the glomeruli. Glomeruli were isolated from GN, normal and control rats and were cultivated in RPMI medium. In normal and control rat cultures, epithelial and mesangial cells were observed. In GN rat cultures, not only epithelial and mesangial cells, but also endothelial and macrophagic cells were identified; the outgrowth capacity of the mesangial cells was enhanced. These data were particularly evident in cultures of GN glomeruli isolated within 2–4 days after the induction of the renal disease, exactly when the glomeruli were infiltrated by a large number of mononuclear phagocytes. It is suggested that the mononuclear phagocytes infiltrating the glomeruli of rats with this model of GN stimulate the proliferation of endothelial and mesangial cells in vitro.     

激活素A在人皮肤微血管内皮细胞中的表达及对其增殖的抑制作用

目的将真核表达质粒pEGFP-AetivinA以脂质体介导转染人皮肤微血管内皮细胞,观察能否在细胞内表达,为进一步研究其作用奠定基础。方法pEGFP-AetivinA扩增和酶切鉴定;原代培养人皮肤微血管内皮细胞,以脂质体介导将真核表达质粒pEGFP-AetivinA转染该细胞,于一定时间内在荧光显微镜下观察绿色荧光蛋白的表达,MTT法检测基因转染对人皮肤微血管内皮细胞增殖的影响。结果转染后48h在荧光显微镜下人皮肤微血管内皮细胞可见发出绿色荧光,72h发出绿色荧光的细胞增多,强度增强;转染后48h与72h,转染组与对照组比较细胞增殖有明显的抑制,差异有显著性（P〈0．05）。结论脂质体可介导pEGFP-AetivinA转染人皮肤微血管内皮细胞,并在细胞内有较高表达,pEGFP-AetivinA基因转染抑制了人皮肤微血管内皮细胞的增殖。     

山莨菪碱和蜕皮激素对内毒素致体外内皮细胞能量代谢的保护作用

目的：探讨山莨菪碱（６５４－２）和蜕皮激素９ＥＤＳ）对内毒素（ＬＰＳ）致伤培养脐静脉内皮细胞（ＨＵＶＥＣｓ）能量代谢的保护作用。方法：采用反相市郊和液相色谱分析法测定ＬＰＳ损伤后培养ＨＵＶＥＣｓ ＡＴＰ、ＡＤＰ、ＡＭＰ及能量负荷（ＥＣ）的变化以及６５４－２、ＥＤＳ的保护作用。结果：ＬＰＳ与内皮细胞孵育后低浓度、早期内皮细胞被激活,ＬＰＳ同一浓度刺激后,随刺激时间延长,ＨＵＶＥＣｓ ＥＣ降低,与正常