VEGF stimulation of endothelial cells induces expression of E-selectin and enhances SDF-1–induced CD34⁺ cell transendothelial cell migration. (A) Confluent monolayers of either BMEC or HUVEC in serum-free conditions were incubated with recombinant endotoxin-free VEGF (40 ng/mL). After an incubation period of 16 hours, the level of E-selectin expression was analyzed by FACS analysis. VEGF upregulated E-selectin by BMEC monolayers (N = 5). Similar results were obtained for HUVEC monolayers (data not shown). IL-1β–stimulated endothelium served as the positive control. (B) Blocking MoAb to E-selectin inhibited SDF-1–induced transmigration of CD34⁺ cells through VEGF-activated endothelial cells. To determine if the E-selectin expression induced by VEGF supported the SDF-1–induced migration, CB-derived CD34⁺ cells were plated on VEGF-primed BMEC monolayers. In the absence of any stimulation to the endothelial monolayers, SDF-1 induced migration of only 1.5% ± 0.2% of the added CD34⁺ cells. SDF-1 induced migration of 15.5% ± 1.1% of CD34⁺ cells through VEGF-activated endothelial monolayers. Blocking MoAb to E-selectin (10 μg/mL) inhibited this migration of CD34⁺cells by 31.6% ± 1.6% (N = 3, P < .05). In addition, SDF-1 induced migration of 22% ± 2.8% of CD34⁺ cells through IL-1β–activated endothelial cells (N = 3, P < .05), which was inhibited by MoAb to E-selectin by 36.0% ± 2.8%. When the endothelial monolayers were treated with both VEGF and IL-1β, SDF-1 induced the migration of 24.8% ± 1.1% of CD34⁺ cells through the endothelial monolayer; MoAb to E-selectin inhibited this migration by 40.4% ± 0.4%.