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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:121.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Microarray-Based Characterization of a Colony Assay Used to Investigate Endothelial Progenitor Cells and Relevance to Endothelial Function in Humans

Aditi Desai; Alexander Glaser; Delong Liu; Nalini Raghavachari; Arnon Blum; Gloria Zalos; Margaret Lippincott; J. Philip McCoy; Peter J. Munson; Michael A. Solomon; Robert L. Danner; Richard O. Cannon, III

From the Translational Medicine Branch (A.D., A.G., A.B., G.Z., M.L., M.A.S., R.O.C.), Genomics Core (D.L., N.R., P.J.M.), and Flow Cytometry Core (J.P.M.), National Heart, Lung, and Blood Institute; and the Functional Genomics and Proteomic Facility (N.R., M.A.S., R.L.D.), Critical Care Medicine Department, NIH Clinical Center; National Institutes of Health, Bethesda, Md.

Correspondence Richard O. Cannon III, MD, National Institutes of Health, Building 10-CRC Room 5-3330, 10 Center Drive, Bethesda, MD 20892-1454. E-mail cannonr{at}nih.gov

Objective— An assay proposed to quantify endothelial progenitor cell (EPC) colonies in humans was investigated to determine the phenotype of recovered cells and their relevance to in vivo endothelial function.

Methods and Results— Twelve sedentary subjects participating in a worksite wellness program underwent endothelial flow-mediated dilation (FMD) testing of the brachial artery and blood sampling for EPC colony assay. Microarray-based genotypic characterization of colonies showed surface markers consistent with T lymphocyte phenotype, but not with an EPC (CD34, CD133, VEGFR-2) or endothelial (CD146) phenotype. Gene expression patterns more closely matched T lymphocytes (r=0.87) than endothelial cells (r=0.66) in our microarray database. Flow cytometry of colonies confirmed large populations of CD3+CD45+ T cells (>75%) and few CD146+CD45– endothelial cells (<1%). Further, there was no correlation between colony number and the magnitude of FMD (r=–0.1512, P=0.6389). After exercise training, subjects improved FMD, from 6.7±2.0 to 8.7±1.9% (P=0.0043). Colonies also increased (P=0.0210), but without relation to FMD (r=0.1074, P=0.7396). T lymphocyte phenotype persisted after exercise (r=0.87).

Conclusions— Cells in a commonly used EPC colony assay have a gene expression and cell surface marker profile consistent with a predominance of T lymphocytes and have an unclear relevance to endothelial function, either before or after exercise training.

Sedentary subjects underwent endothelial testing and blood sampling at baseline and after 3 months of exercise training. Microarray and flow cytometry-based characterization of colonies from an assay proposed to quantify endothelial progenitor cells was consistent with T lymphocytes, but not endothelial cells. After exercise training, subjects improved endothelial function (P=0.0043) and colony number (P=0.0210), but T lymphocyte phenotype persisted.

Key Words: exercise • gene expression • endothelial function • endothelial progenitor cell • T lymphocyte

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