Cytofluorometric detection of human endothelial cells in whole blood using S-Endo 1 monoclonal antibody

George, F.; Poncelet, Pascal; Laurent, Jean-Claude; Massot, O.; Arnoux, D.; Lequeux, N; Ambrosi, Pı̈erre; Chicheportiche, C.; Sampol, José

doi:10.1016/0022-1759(91)90352-g

Cited by 70 publications

(42 citation statements)

References 24 publications

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“…Furthermore, the maturation process of ECs is not clearly understood, so the use of various marker combinations is necessary. The endothelial marker CD146, which is often used in EC analyses, is also expressed on active T lymphocytes [8,31]. The surface marker, CD144 (VE-cadherin), is expressed on both ECs and fetal hematopoietic stem cells [20,32,33].…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric evaluation of circulating endothelial cells: A new protocol for identifying endothelial cells at several stages of differentiation

et al. 2007

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Several factors may influence the analysis of endothelial cells (ECs) by flow cytometry: separation of mononuclear cell, washing and centrifugation steps, panel of monoclonal antibodies, and the lack of standardization of gating technique. Therefore, the reliable quantification of ECs remains a technical challenge. The purpose of this study is to define a new flow cytometric protocol to characterize and quantitate ECs. In previous investigations, increased numbers of circulating ECs have been found in sickle cell disease. The patients with sickle cell disease might provide useful material for the study. We performed flow cytometry on whole blood from 20 normal controls and 31 patients with sickle cell disease (20 patients with steadystate disease and 11 patients with vaso-occlusive crises) using a lyse/no-wash procedure, specific and nonspecific antibody combinations (CD146, CD144, CD34, and CD117), and broad gating. This protocol produced much higher values for the number of circulating ECs (a mean of 2,396.55 ± 658.37 ECs/mL in controls vs 6,709.60 ± 1,772.32 ECs/mL in the steady-state group, or 18,213.50 ± 8,451 in the vaso-occlusive crises group, P < 0.001 for both), and also showed variable EC size and granularity, which may reflect activated, or early release ECs. This novel protocol performed comparably in terms of reproducibility, reliability, and dilution linearity with a previously described protocol. This approach has significant advantages for the characterization and quantitation ECs compatible with the pathophysiology. Using the specific antibodies, CD146 and CD144, together may give more informative EC data than the general approach used. Am. J. Hematol. 82:706-711, 2007. V

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Section: Discussionmentioning

confidence: 99%

Flow cytometric evaluation of circulating endothelial cells: A new protocol for identifying endothelial cells at several stages of differentiation

et al. 2007

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“…26 The other mAbs used were anti-PECAM-1 mAb (mouse IgG1; Novocastra Laboratories, Newcastle, United Kingdom), anti-cadherin-5 mAb (mouse IgG1; Immunotech, Marseille, France), anti-major histocompatibility complex (MHC) class I (mouse IgG1; Immunotech), anti-VE-cadherin polyclonal antibody (D. Gulino, Grenoble, France), antitransferrin receptor (A. Le Bivic, Marseille, France), antiplacental alkaline phosphatase (Dako, Trappes, France), ST4 and goat anti-rabbit IgG fractions coupled with 10 nm colloidal gold (GAR-10) were purchased from British Biocell (Cardiff, United Kingdom).…”

Section: Methodsmentioning

confidence: 99%

Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion

Bardin

Anfosso

Massé

et al. 2001

Blood

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CD146 is a cell-surface molecule belonging to the immunoglobulin superfamily and expressed in all types of human endothelial cells. Confocal and electron microscopic analysis of confluent human umbilical vein endothelial cells (HUVECs) were used to demonstrate that CD146 is a component of the endothelial junction. Double immunolabeling with vascular endothelial cadherin showed that CD146 is localized outside the adherens junction.Moreover, CD146 expression is not restricted to the junction, since part of the labeling was detectable at the apical side of the HUVECs. Interestingly, cell-surface expression of CD146 increased when HUVECs reached confluence. In addition, the paracellular permeability of CD146-transfected fibroblast cells was decreased compared with that of control cells. Finally, CD146 colocalized with actin, was partly resistant to Triton X-100 extraction, and had its expression altered by actindisrupting agents, indicating that CD146 is associated with the actin cytoskeleton. These results show the regulated expression of CD146 at areas of cell-cell junction and strongly suggest involvement of CD146 as a mediator of cell-cell interaction. ( IntroductionThe vascular endothelium forms a continuous monolayer on the inner surface of the vessel wall and plays a pivotal role in regulating blood flow, vascular permeability, thrombogenesis, and hematogenous metastasis. 1 Positioned at the interface between blood and tissues, quiescent endothelial cells (ECs) generate an antithrombotic surface equipped to respond quickly to biologic needs. 2 The endothelial monolayer requires highly effective intercellular junctions that control the contact between adjacent cells and the trafficking of circulating blood cells. 3,4 At least 2 types of cell-cell junctional structures have been identified in the endothelium: adherens junctions (AJs) and tight junctions (TJs). These play a central part in the control of paracellular permeability and maintenance of cell polarity. [5][6][7] The junctions are tightly regulated structures composed of several adhesion molecules interacting with cytoskeletal proteins. Among the adhesive molecules, the endothelium-specific cadherin 5 or vascular endothelial cadherin (VEcadherin) 8,9 is localized in AJs, whereas the junctional adhesion molecule (JAM) 10 was reported to be present in TJs. Other molecules, such as platelet endothelial cell adhesion molecule 1 (PECAM-1)/CD31, are not restricted to one type of junctional structure, and their specific localization appears to be important to their vascular functions. 11,12 The S-Endo 1-associated antigen (CD146), also referred to as MelCAM or MUC18, 13 is a transmembrane glycoprotein that is constitutively expressed in the whole human endothelium, irrespective of its anatomical site or vessel caliber. 14,15 CD146 expression is not restricted to ECs; it has also been observed on several other cell types, including melanoma cells, 13 smooth muscle cells, and follicular dendritic cells. 14 Using optical microscopy, we previously showed that,...

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“…S-endo1 strongly reacted with the majority of human endothelium from different types of vessels, including arteries, arterioles, veins, veinules, capillaries, high endothelial venules, and lymphatic vasculature. (16) However, MUC 18 staining was restricted on endothelium to capillaries and high endothelial venules. (25) Moreover, S-endo1 strongly reacted with vessel of umbilical cord in frozen cryostat sections whereas MUC 18 did not, regardless of identical antigen of the two monoclonal antibodies (MAb).…”

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confidence: 99%

“…(9) Also, CD146 has been proven to be expressed on circulating endothelial cells as a maker of endothelium. (12)(13)(14) Two monoclonal antibodies, S-endo1 (15,16) and P1H12, (17) which are demonstrated to recognize CD146, had been developed to detect circulating endothelial cells in ANCA-associated small-vessel vasculitis. Both of them can bind to the endothelia of various tissue sections and different types of vessels, indicating the widespread expression of CD146 in endothelium.…”

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confidence: 99%

Generation and Characterization of a Panel of Monoclonal Antibodies Against Distinct Epitopes of Human CD146

et al. 2008

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CD146 (MUC18, Mel-CAM/MCAM) is a transmembrane protein, originally identified as a biomarker of melanoma, and plays an important role in cancer invasion and metastasis. Further studies revealed that CD146 as a novel endothelial marker was also involved in angiogenesis. Previous studies reported several anti-CD146 antibodies, such as MUC18, A32, S-endo1, and P1H12, showing different binding patterns to the endothelium of various types of blood vessels. To examine the possibility that antibodies targeting different epitopes on CD146 could have different behaviors, we generated a panel of anti-human CD146 monoclonal antibodies, named AA1-5 and AA7, by immunizing mice with human CD146 protein purified from HUVEC. Their specificity and binding affinity were intensively characterized using Western blotting, flow cytometry, and immunohistochemical assay. On the basis of epitope mapping, we divided the six monoclonal antibodies (MAb) into two groups, groups V1 and C2-2, corresponding to the different extracellular domains harboring these epitopes, the first IgV and the second IgC2 domains, respectively. Furthermore, owing to different epitopes, the two groups of antibodies behaved differentially in cellular and histological levels. Therefore, these anti-CD146 MAbs targeting different domains should be useful tools in studying the expression and function of human CD146. 345

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Cytofluorometric detection of human endothelial cells in whole blood using S-Endo 1 monoclonal antibody

Cited by 70 publications

References 24 publications

Flow cytometric evaluation of circulating endothelial cells: A new protocol for identifying endothelial cells at several stages of differentiation

Flow cytometric evaluation of circulating endothelial cells: A new protocol for identifying endothelial cells at several stages of differentiation

Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion

Generation and Characterization of a Panel of Monoclonal Antibodies Against Distinct Epitopes of Human CD146

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