Characterization of Endothelial Progenitor Cells in Healthy Subjects: Effects of Different Factors on Immunophenotype and Clonogenic Potential.

There are experimental evidences that Endothelial Progenitor Cells (EPCs) is a pool of cells that could proliferate and differentiate into mature endothelial cells and that may contribute to endothelial repair, but no systematic studies exist regarding physiologic variations in EP circulating pool and in CFU-Endo capacity in healthy subjects. In this regard, since the level of EPCs may be a biologic marker for vascular function, we decided to characterize primitive and circulating EPCs and their relation with gender, aging and the capacity to form CFU-Endo in a selected healthy population. Each subject underwent a screening evaluation to exclude any underlying cardiac diseases. A 20mL sample of peripheral blood was obtained (h 11:00–12:00 AM) from 15 male and 17 female age matched subjects (mean age 41.6±15.9yrs). EPCs were assessed by flow cytometric analysis on 100,000 events per sample (FACScan, Becton Dickinson). Primitive EPCs, defined as CD45−/34+/133+ and CD45−/KDR+/144−, were 8±1.8SE/uL. Circulating EPCs, defined as CD45−/146+/31+/FCS^low and CD45−/146+/144+, were 18.72±2.5SE/uL. No correlation was found between these populations and aging, gender and BMI. Instead a significant decrease in CD45−/34+/133+ (2.9±0.6SE/uL) subpopulation was observed with aging (r = 0.45; p = 0.009). CFU-Endo were performed as described by Hill JM et al. (NEJM 2003) and scored by microscopy in blind by two operators. The confirmation of endothelial-cell lineage was performed by immunostaining for endothelial-specific antibodies (CD31+, CD105+, CD144+, CD146^low, CD133−, vWf-). CFU-Endo were expressed as the mean number of colonies per well in 8–25 separate determinations for each subjects and only 15 subjects gave colonies (mean 2.1±0.05SE). CFU-Endo do not correlate with aging and gender in healthy subjects confirming that endothelial injuries and cardiovascular risk factors are a crucial endothelium stimulus. In fact, recently it has been reported that higher colony counts were found in subjects with various degrees of cardiovascular risks. In conclusion CD45-/34+/133+ cell population correlates with aging but since this population represents Hemato-Endo Precursors that include a majority of endothelial progenitors and a small number of hematopoietic stem cells, further studies will be needed to identify a subpopulation strongly predictive of CFU-Endo capacity and to assess its endothelial lineage switch.