Autocrine effects of VEGF-A on the adherence of Monomac6 (MM6) cells to HUVECs. (A) Serum-starved HUVECs were treated for 24 hours with VEGF-A (100 ng/mL) and perfused with Monomac 6 cells treated with a blocking anti–VLA-4 antibody, sPECAM-1.Fc, or sJAM.Fc (10 μg/mL, 30 min). Some HUVECs were treated with anti–ICAM-1 Ab prior to perfusion. Cell arrest and spreading were analyzed in multiple high-power fields recorded by video microscopy. Cells not moving for more than 30 seconds were defined as adherent. VEGF-A significantly induced the adherence of MM6 cells at a shear rate of 1.5 dyne/cm². Values represent mean ± SEM of 3 to 5 experiments performed in triplicate; *P < .05 versus control and **P < .05 versus VEGF-A. (B) Immunofluorescence microscopy of HUVECs treated with VEGF-A (10 ng/mL) for 12 hours after staining for endothelial cell marker PECAM-1 and VCAM-1. Stimulation with VEGF-A induces redistribution of constitutively expressed PECAM-1 to the apical side of the endothelial cells. No constitutive expression of VCAM-1 is observed; VCAM-1 expression is induced by VEGF-A exposure. (C) Quantitative real-time reverse-transcription–polymerase chain reaction analysis of total RNA isolated from HUVECs confirmed that PECAM-1 expression is not changed after 6- and 12-hour VEGF treatment. The expression of VCAM-1 mRNA is significantly up-regulated, and that of ICAM-1 significantly down-regulated. MCP-1 mRNA expression level is almost completely inhibited by VEGF-A. Levels are normalized to that of hHRPT as well as cyclophilin A mRNA. *P < .05 versus control. Error bars indicate SD.