Comparative Analysis of Methods for Assessment of Circulating Endothelial Progenitor Cells

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Abstract
The number and properties of endothelial progenitor cells (EPC) in disease states is of considerable interest due to the importance attributed to this distinct cell population. However, there has been no study comparing each of the methods employed in the same sampled individuals. Herein, we performed an analysis of several methods used for circulating EPC assessment and correlated them with humoral factors known to influence their numbers. Thirty-eight individuals (mean age of 34 ± 9 years) were tested. Peripheral blood mononuclear cells were obtained and stained for FACS analysis with antibodies to CD34, CD45, CD133, and KDR and the remaining cells grown under endothelial cell conditions for assessment of colony-forming unit (CFU) numbers and adhesive properties. Levels of circulating vascular endothelial growth factor (VEGF), erythropoietin (EPO), and C-reactive protein (CRP) were determined and correlated with each of the EPC markers. CFU numbers did not correlate with CD34/KDR or CD34/CD133/KDR and negatively correlated with CD34/ CD133 numbers. CD34/KDR numbers correlated with CD34/CD133/KDR, but not with CD34/ CD133. Only CD34/KDR and CD34/CD133/KDR correlated with VEGF serum levels. The number of EPC adhering to fibronectin and endothelial cells correlated with CFU numbers and not with either of the EPC membrane markers. Current methods for quantitatively assessing numbers of circulating EPC are not correlated. VEGF serum levels are associated only with CD34/KDR and CD34/ CD133/KDR, whereas CFU numbers correlate with EPC functional properties. These findings may suggest that CD34/KDR is more appropriate for the definition of circulating EPC, whereas CFU numbers are more likely to reflect their ability to proliferate.