Comparative Analysis of Methods for Assessment of Circulating Endothelial Progenitor Cells Indicated Heterogeneity of Subpopulations at Various Stages of Differentiation.

The cells of the vasculature are derived from pluripotent stem cells, known as hemangioblasts, which also give rise to blood. Angiogenesis, the formation of new blood vessels is considered to result from the proliferation and migration of mature endothelial cells from existing blood vessels or from the recently described, but not yet well characterized endothelial progenitor cells (EPCs). The number and properties of EPC in disease states is of considerable interest due to the promising therapeutic potential of these cells. EPCs have been shown to be mobilized from the bone marrow and contribute to angiogenesis following vascular injury, organ ischaemia and tumor progression. However, mechanisms that drive the EPC response to injury remain elusive and the lack of definitions of EPC subpopulations and the many methods used by different groups to identify these cells make correlation of results difficult. To begin to understand the potential of these cells, we performed a comparative analysis of several methods used for circulating EPC assessment in 40 healthy individuals (mean age of 33±9 years) and correlated them with humoral factors known to influence their numbers. Peripheral blood mononuclear cells were obtained and evaluated by flow cytometeric 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 and C-reactive protein were determined and correlated with each of the EPC markers. CFU numbers did not correlate with the number of CD34/KDR (VEGFR2) or CD34/CD133/KDR positive cells and negatively correlated with CD34/CD133 (which includes hematopoietic progenitors). CD34/KDR number correlated with CD34/CD133/KDR but not with CD34/CD133. Only the VEGFR2 positive cells populations (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 but not with EPC membrane markers. Our results indicate for the first time that EPC, like hematopoietic precursors may be a heterogeneous cell population comprised of progenitors at various stages of differentiation, having varied proliferative capacity. This could explain the lack of correlation in results obtained using different methods for quantitatively assessing the numbers of circulating EPC. These data also suggest that co-expression of CD34, CD133and KDR-VEGFR2 is appropriate for defining a population of circulating EPC whereas CFU are more likely to reflect the proliferative capacity of the progenitors.