PTN and M-CSF together induce VEC gene expression in fresh monocytes in vitro. Human CD14⁺ monocytes were purified from PB by density gradient centrifugation and anti-CD14 antibody treatment with magnetic bead selection. After 1 hour of culture, M-CSF (10 ng/mL) was added, and/or PTN (50 nM) and/or VEGF (20 ng/mL) were added twice to the cultures (after 24 hours and 5 days of culture). Cells were cultured for a total of 7 days. Total RNA was isolated from 10⁶ cells containing monocytes treated with various cytokine combinations that were serially diluted with T cells. RT-PCR was performed with primers capable of detecting VEC gene expression (Tie-2, Flk-1, VWF, and VE-Cad [cadherin]) with a sensitivity of 1 human coronary arterial endothelial cell (HCAEC) in 10⁶ T cells that lacked VEC gene expression to show that there was no VEC contamination within the purified fresh monocyte population. GAPDH was used as a control. Using primers for the monocyte-specific gene c-fms, monocytes were detected with a sensitivity of 1 cell diluted in 10⁶ T cells.