CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs)

Endothelial colony-forming cells (ECFC) are grown from circulating CD34⁺ progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34⁺ and CD34⁻ ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34⁺ and CD34⁻ ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34⁺: 95 % pos; CD34⁻: 99 % neg). Both fractions proliferated at same rate, while CD34⁺ ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3⁴⁻ cells. However, during cell culture CD34⁻ cells re-expressed CD34. Cell-seeding density, cell–cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell–cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34⁺ and CD34⁻ cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function.