title: Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype.
abstract: Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.
authors: Margaret M
authors: MM
authors: Tropea
authors: Bonnie J A
authors: BJ
authors: Harper
authors: Grace M
authors: GM
authors: Graninger
authors: Terry M
authors: TM
authors: Phillips
authors: Gabriela
authors: G
authors: Ferreyra
authors: Howard S
authors: HS
authors: Mostowski
authors: Robert L
authors: RL
authors: Danner
authors: Anthony F
authors: AF
authors: Suffredini
authors: Michael A. Solomon, MD, Critical Care Medicine Department, Clinical Center, National Institutes of Health, Building 10, Room 2C145, Bethesda, MD 20892-1662, USA, Tel.: +1 301 496 9320, Fax: +1 301 402 1213, E-mail: msolomon@cc.nih.gov.
authors: Michael A
authors: MA
authors: Solomon
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000378:metabolism*; D051929:CD146 Antigen / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D002470:Cell Survival; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000097:blood; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008853:Microscopy; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D010641:Phenotype; D010865:Pilot Projects; D037121:Plant Lectins / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D051929:CD146 Antigen; D007211:Indoles; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C036850:Ulex europaeus lectins; D014841:von Willebrand Factor; D003609:Dactinomycin; C007293:DAPI; D012313:RNA; C025942:7-aminoactinomycin D; D017493:Leukocyte Common Antigens
keywords: Vascular cell markers; circulating endothelial cells; flow cytometry; gene expression; viability