Comparative Analysis of Methods for Assessment of Circulating Endothelial Progenitor Cells

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.

Reduction of both number and proliferative activity of human endothelial progenitor cells in obesity

“…Homologous results for all obesity-associated parameters tested are found for CD34 þ EPCs, that is, CD34 þ /CD133 þ / KDR þ triple-and CD34 þ /KDR þ double-positive EPCs. These results underline an earlier observation that exactly these two phenotypes are most closely related to the levels of vascular endothelial growth factor (VEGF) in vivo 31 and are therefore highly consistent with the definition of circulating EPCs. Nevertheless, even CD133 þ /KDR þ EPCs and CFUs reflect the tendencies found for CD34 þ EPCs with high probability.…”

“…30 Although CFUs poorly mirror circulating numbers of EPCs, they have been assumed to reflect the ability of EPCs to proliferate. 31 By daily documented microscopic monitoring of the plated PBMCs, we noticed that the outgrowing cells of the obese group showed delayed proliferation compared with those of the lean group. These findings were confirmed in pilot experiments using the general proliferation marker PCNA and led us to a subsequent detailed evaluation of cell-cycle proteins.…”

Level of CD14+-endothelial progenitor cells is not associated with coronary artery disease or cardiovascular risk factors

“…First, George et al (2006) found that, in healthy individuals, there was no correlation between the various methods used for estimating EPC numbers. Thus, there are fundamental methodological differences that likely account for the different results.…”

“…Alternatively, late EPCs possess high proliferation potency and express CD34 (Hur et al 2004;Lin et al 2000;Reyes et al 2002). In addition, ex vivo cultivation procedures may confound the influence of cardiac risk factors on the number of EPCs, whereas the direct measurement of EPCs might more closely resemble in vivo conditions (George et al 2006). In this study, we directly determined the number of CD14 + KDR + cells, which correspond to early EPCs, in peripheral blood using flow cytometry.…”

Circulating Endothelial Progenitor Cells as a New Marker of Endothelial Dysfunction or Repair in Acute Stroke

“…Emerging evidence indicates that CFU number more appropriately represents EPC functional properties rather than EPC number. 1,2,15 Whereas EPC function is critical for endothelial maintenance and repair, CFU number may indicate a cumulative vascular risk in ischemia. On the other hand, EPCs constitute a heterogenous population with distinct growth characteristics.…”