Measuring angiogenic cytokines, circulating endothelial cells, and endothelial progenitor cells in peripheral blood and cord blood: VEGF and CXCL12 correlate with the number of circulating endothelial progenitor cells in peripheral blood

Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) are thought to play an important role in the vascularization of damaged tissues and cancers. These cells are also required for tissue-engineered blood vessels and to help skin substitutes revascularize more efficiently. A standard approach to the phenotyping and enumeration of CEC and EPC is key to the development of new therapies, and the identification of biomarkers within the blood that regulate their levels may be important for the treatment of cancer. We have devised an improved multiparameter flow cytometric assay for CEC and circulating EPC enumeration. This assay uses antibodies recognizing CD133 and CD34 to identify EPC and CEC, respectively, and incorporates specific markers CD144 and vascular endothelial growth factor receptor 2 (VEGFR-2) for both CEC and EPC cells. In peripheral blood (PB), mean CEC numbers were 55 +/- 95 mL(-1) and mean EPC numbers were 44 +/- 58 mL(-1) (n = 60). We also found a significant correlation of both plasma VEGF (r = 0.90, p < 0.001) and CXCL12 (r = 0.84, p < 0.001) with EPCs, but not CECs. The cytokines also correlated with each other (r = 0.85, p < 0.001). In umbilical cord blood (UCB) we found on average 13 times more CEC (719 +/- 338 mL(-1)) and 7 times more EPC (299 +/- 245 mL(-1)) than in PB. However, serum VEGF and CXCL12 levels in UCB did not correlate with either EPC or CEC numbers. These results suggest a major role for VEGF and CXCL12 in the control of marrow-derived EPCs in adult PB and provide normal data for comparison with patient populations.     

A novel flow cytometric approach to distinguish circulating endothelial cells from endothelial microparticles: relevance for the evaluation of endothelial dysfunction

Circulating endothelial cells (CEC) and endothelial microparticles (EMP) are emerging as markers of endothelial repair and activation/apoptosis. Although significant changes in the number of CEC and EMP in pathological conditions have been reported, their reliable identification and quantification still remain a technical challenge. Here, we present a novel methodology for the identification and quantitation of CEC and EMP based on multicolor flow cytometry. Using a lyse/no wash protocol, we observed that in 50 μl of peripheral blood, the large majority of events expressing an endothelial phenotype (CD45-/CD146+/CD34+) are due to non-nucleated particles (DRAQ5-) carrying mitochondrial activity (MitoTracker+) and, therefore, classified as EMP. We enumerated circulating EMP by single platform absolute count in a lyse/no wash four-color flow-cytometric procedure, which allowed the distinction, within the whole endothelial compartment, of EMP derived from endothelial progenitors (CD45-/CD146+/CD34+/CD117+) and from mature endothelial cells (CD45-/CD146+/CD34+/CD117-). A significant increase in both subsets was observed in patients with diabetes mellitus. Thus, this simple and highly reproducible method may be useful for monitoring endothelial dysfunction in clinical settings.     

急性心肌梗塞患者外周血中循环内皮细胞含量的改变及凋亡的检测

目的应用我所新建立的循环内皮细胞（Ｃｉｒｃｕｌａｔｉｎｇ Ｅｎｄｏｔｈｅｌｉａｌ Ｃｅｌｌｓ，ＣＥＣ）的分离计数方法计数急性心肌梗塞患者外周血中循环内皮细胞含量的改变并检测其凋亡。方法Ｐｅｒｃｏｌｌ密度梯度离心后，将其先后标记以鼠抗人ＣＤ３１抗体，ＦＩＴＣ－羊抗鼠ＩｇＧ及碘化丙啶。应用流式细胞伙计数ＣＤ３１阳性细胞的百分比，并通过检测ＣＥＣ的ＤＮＡ倍体含量检测其凋亡状态。结果急性心肌梗塞组（ｎ＝１６）与正常对照组（ｎ＝１６）的 ＣＥＣ分别为 ６２．９±２４．５／ｍｌ及 ２９． ９±９．１／ｍｌ全血，两者有非常显著性差异（Ｐ<０．０００１）。两组均为双倍体ＤＮＡ，无亚二倍体峰的出现，说明无细胞凋亡的发生。结论此方法克服了以往ＣＥＣ研究方法的缺点，提高了特异性及回收率，使ＣＥＣ真正成为来自活体的特异的且直接反映血管损伤的量化指示物。     

Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules

Endothelial cells express a wide spectrum of surface molecules involved in multiple vascular functions. We quantitatively determined an extensive immunologic phenotype of endothelial cells through a large panel of antibodies directed against i) well-known endothelial molecules CD31, CD34, CD49b, e, f, CD51, CD54, CD55, CD62E and P, CD105, CD106, HLA class I and HLA class II; ii) molecules defined by monoclonal antibodies newly clustered during the 6th workshop of Human Leukocyte Differentiation Antigen (HLDA) CD109, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146 and CD147; iii) molecules defined by unclustered monoclonal antibodies. The expression of these molecules was quantified on human umbilical vein endothelial cells (HUVEC) cultured in resting conditions and after stimulation with IL-ip (10 U/ml), TNF-a (10 ng/ml) and phorbol myristate acetate (60 ng/ml). Some molecules were constitutively expressed, and others were negative, which served to determine the basal phenotype. After cell stimulation, the molecules showed weak or strong expression modulation, leading to the definition of an activated phenotype. Changes in the kinetics and the amplitude of expression served to characterize poorly defined molecules and may be useful to determine their physiologic role. Also, we compared the phenotypes of endothelial cell lines EA.hy 926 and ECV 304 to that of HUVEC to assess their reliability as an endothelial cell model. Each cell line displayed a specific repertoire of molecules expressed at different levels, which could have significant im-I plications for cell line behavior as endothelial cells.     

Hematopoietic engraftment in recipients of unrelated donor umbilical cord blood is affected by the CD34+ and CD8+ cell doses.

Umbilical cord blood (UCB) transplantation is limited by the low number of hematopoietic stem cells in UCB units, which results in a low engraftment rate in transplant recipients. Here, we measured the total nucleated cell count and CD34(+), CD3(+), CD4(+), CD8(+), CD14(+), and CD16(+)/56(+) cell doses in each UCB unit and evaluated their influence on engraftment and other outcomes in 146 recipients. Multivariate analysis showed a significant association between a higher incidence of successful engraftment and a dose of CD34(+) and CD8(+) cells above the median (1.4 x 10(5) and 15.7 x 10(5) cells/kg, respectively). Engraftment occurred 4 days earlier in patients who received UCB with more than the median dose of CD34(+) cells than those receiving UCB at or below the median. Stratification of the group according to CD34(+) cell dose revealed a significant influence of the CD8(+) cell dose on the time to achieve neutrophil engraftment in patients receiving a lower CD34(+) cell dose, whereas there was no significant influence in the patients receiving a higher CD34(+) cell dose. These results suggest that consideration of CD34(+) and CD8(+) cell doses in UCB units may improve the engraftment in recipients of UCB transplantation.     

Activated T lymphocytes in pre-eclampsia

PROBLEM: The aim of our study was to investigate the activation markets of T CD3(+), T helper CD4(+) and T cytotoxic CD8(+) cells, as well as, the populations of T na?ve CD4(+) CD45RA(+), T memory CD4(+) CD45RO(+) and T regulatory lymphocytes in PE and healthy pregnant women. METHOD OF STUDY: Twenty-five patients with PE and thirty healthy third trimester pregnant women were included in the study. Peripheral blood mononuclear cells were isolated from peripheral blood, stained with monoclonal antibodies and estimated using the flow cytometric method. RESULTS: The percentages of CD4(+)CD25(+), CD4(+)CD25(dim), CD3(+)HLA-DR(+), CD4(+)HLA-DR(+) and CD8(+)HLA-DR(+) cells did not differ between study groups. The population of T regulatory CD4(+)CD25(bright) lymphocytes was significantly lower in the group of patients with PE when compared with the controls (P < 0.01). The percentages of CD3(+)CD25(+) (P < 0.05), CD8(+)CD25(+) (P < 0.05), CD4(+)45RO(+) (P < 0.01) lymphocytes were significantly higher, while CD4(+)CD45RA(+) (P < 0.01) cells--significantly lower in peripheral blood of patients with PE when compared with the control group. CONCLUSION: The increased levels of T CD4(+)45RO(+) and T CD8(+) CD25(+) cells can suggest the activation of CD4(+) and CD8(+) T lymphocytes in pre-eclampsia. It seems possible that the activation of T lymphocytes is associated with the deficiency of T regulatory cells in PE.     

内皮祖细胞的体外培养

 目的 建立一个体外培养脐血来源内皮祖细胞（EPC）的培养体系。方法 脐带血经密度梯度离心获得单个核细胞,按本室已建立的培养体系细胞培养,免疫细胞化学和流式细胞术对培养7d后的细胞进行CD34、CD133、vWF、CD146及CD144鉴定。结果 接种后前5d为生长的潜伏期,细胞开始贴壁,无明显扩增。第6天平均每个视野下细胞数目为287+45;第9天细胞数为282+46;第12天开始,细胞进入对数生长期,细胞数为805+67（P<0.05）;第19天细胞继续增殖,细胞数为1115+182（P<0.05）;第23天时,细胞进入凋亡期,数量明显减少,为265+61（P<0.05）。vWF,CD146,CD144表达阳性。流式细胞术结果表明,梭形样细胞群体中,CD34阳性率为88.98%+5.15% (P<0.05),CD133阳性率为1.20%+1.44% (P<0.05)。结论 利用本实验室的培养体系成功培养出内皮祖细胞。     

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUNT beads and the whole blood "Lyse/No-Wash" protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 microl of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 h after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained.     

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 micro g/ml for 0-60 min at 37 degrees C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 micro g/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.