VAP-1 supports PMN transmigration under flow conditions. (A) Phase-contrast micrographs showing surface-adherent (phase-bright; 2 pointed out by open arrows) and transmigrated (phase-dark; 3 indicated by thin white arrows) leukocytes. Numbers 1 and 2 indicate 2 representative endothelial cells at the bottom of the capillary. The first frame is taken 5 minutes after the perfused leukocytes appeared in the microscopic field, and the second one was captured 10 minutes later. The area of the microscopic picture is 0.02 mm² (ie, it is 1/15th of a single microscopic field and 15 separate microscopic fields were analyzed for each treatment in each individual experiment). The actual transmigration process can be seen in the Supplemental Video online at the Blood website. Note that the surface-bound cells do not remain stationary during the assay, since they also actively migrate on the HUVEC surface in search of junctions. Bar, 20 μm. (B) The number of transmigrated cells was determined after pretreating VAP-1- or VAP-1Y471F-transfected HUVECs by vehicle and SSAO inhibitors (BTT = BTT-2027, SC + HA = semicarbazide + hydroxylamine). (C) The effect of anti-VAP-1 mAb alone or in combination with SSAO inhibitors on the transmigration was determined as in panel B. All results are mean ± SEM of 4 to 7 independent experiments using PMNs and HUVECs from different individuals. (D) VAP-1 supports PMN transmigration. The number of PMNs transmigrating through HUVECs transfected with VAP-1, enzymatically inactive VAP-1, and lacZ was determined. The results are mean ± SEM of 4 to 5 independent experiments using HUVECs and PMNs from different individuals. The number of PMNs transmigrating through VAP-1 transfectants is defined as 100%, and thus HUVECs transfected with VAP-1 support statistically significantly more transmigration than HUVECs transfected with enzymatically inactive VAP-1 or with lacZ. ^*P < .05; ^**P < .01. Endoth. indicates endothelial cells; pretreat., pretreated; and vehic, vehicle.