Granulocyte transmigration through the endothelium is regulated by the oxidase activity of vascular adhesion protein-1 (VAP-1)‏ by Kaisa Koskinen, Petri J. Vainio, David J. Smith, Marjo Pihlavisto, Seppo Ylä-Herttuala, Sirpa Jalkanen, and Marko Salmi Blood Volume 103(9):3388-3395 May 1, 2004 ©2004 by American Society of Hematology

SSAO activity is important for VAP-1-dependent rolling of PMNs on HUVECs under shear. SSAO activity is important for VAP-1-dependent rolling of PMNs on HUVECs under shear. (A) Two video frames taken 3 seconds apart showing rolling and stably adherent PMNs on VAP-1-transfected HUVECs (t = 0 is by definition the moment when infused leukocytes appear on the endothelial cells in the microscopic field). The open arrows point to 2 rolling cells (nos. 1 and 2; their rolling path during the 3-second interval is indicated by the dotted arrow), and some of the firmly adherent cells are pointed out by thin black arrows (nos. 3-6). The shear was 1.0 dyn/cm2. Bar, 20 μm. (B) The SSAO inhibitors (BTT = BTT-2027, SC + HA = semicarbazide + hydroxylamine) block rolling of PMNs on VAP-1-transfected HUVECs, but have no effect on rolling on VAP-1Y471F-transfected cells. The number of rolling PMNs was counted after each treatment and compared with that seen in the vehicle-treated capillaries. The results are mean ± SEM from 3 to 5 independent experiments using HUVECs and PMNs isolated from different individuals. (C) The effect of anti-VAP-1 mAb on PMNs rolling on VAP-1- and VAP-1Y471F-transfected HUVECs was analyzed as described in panel B in the presence of control (HB116) and anti-VAP-1 (TK8-14) mAbs. The results are mean ± SEM (n = 3-4). *P < .05; **P < .01. Endoth. indicates endothelial cells; pretreat., pretreated; and vehic, vehicle. Kaisa Koskinen et al. Blood 2004;103:3388-3395 ©2004 by American Society of Hematology

VAP-1 supports PMN transmigration under flow conditions. 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. Kaisa Koskinen et al. Blood 2004;103:3388-3395 ©2004 by American Society of Hematology

Expression of VAP-1 in adenovirally transfected HUVECs Expression of VAP-1 in adenovirally transfected HUVECs. Nontransfected HUVECs (native) and cells transfected with the pADENO-lacZ, pADENO-VAP-1, or pADENO-VAP-1Y471F (enzymatically inactive point mutant) were stained with 3G6 (negative control [neg. co.]), ... Expression of VAP-1 in adenovirally transfected HUVECs. Nontransfected HUVECs (native) and cells transfected with the pADENO-lacZ, pADENO-VAP-1, or pADENO-VAP-1Y471F (enzymatically inactive point mutant) were stained with 3G6 (negative control [neg. co.]), HB116 (against HLA class I), and TK8-14 (against VAP-1) and analyzed using FACS. The x-axis is the fluorescence intensity in a log-scale, and the y-axis is the relative number of cells. Kaisa Koskinen et al. Blood 2004;103:3388-3395 ©2004 by American Society of Hematology

Inhibition of SSAO activity by small molecular SSAO inhibitors and by VAP-1Y471F mutation. Inhibition of SSAO activity by small molecular SSAO inhibitors and by VAP-1Y471F mutation. Uninfected (native), pADENO-lacZ-, pADENO-VAP-1-, and pADENO-VAP-1Y471F-infected HUVECs were treated with the vehicle (-) or with SSAO inhibitors (SC + HA = semicarbazide + hydroxylamine, BTT = BTT-2027 compound) and antibodies (HB116 against HLA class I, TK8-14 against VAP-1), and the enzymatic activity was determined using the radiochemical method. The specific activities (mean ± SEM from 3 independent assays) are shown. The SSAO-dependent H2O2 production in HA + SC-treated native HUVECs is 0 by definition. Note that the VAP-1Y471F mutant is devoid of any SSAO activity and that the anti-VAP-1 mAb does not interfere with the SSAO activity. Kaisa Koskinen et al. Blood 2004;103:3388-3395 ©2004 by American Society of Hematology

VAP-1 is not involved in stable adhesion between PMNs and HUVECs VAP-1 is not involved in stable adhesion between PMNs and HUVECs. (A) The SSAO inhibitors and (B) anti-VAP-1 mAbs do not have any effects on firm binding of PMNs to VAP-1-transfected cells stimulated with 5 U/mL TNF-α (data from the rolling assay protocol). VAP-1 is not involved in stable adhesion between PMNs and HUVECs. (A) The SSAO inhibitors and (B) anti-VAP-1 mAbs do not have any effects on firm binding of PMNs to VAP-1-transfected cells stimulated with 5 U/mL TNF-α (data from the rolling assay protocol). (C-D) Stronger inflammatory stimulus increases the number of adherent cells, but does not make the binding VAP-1-dependent (data from the transmigration assay protocol). HUVECs transfected with VAP-1 or VAP-1Y471F were pretreated with the various compounds as indicated in the figure, and the numbers (mean ± SEM; n = 3-7) of firmly adherent cells were determined. None of the effects was statistically significant. Endoth. indicates endothelial cells; pretreat., pretreated; and vehic, vehicle. Kaisa Koskinen et al. Blood 2004;103:3388-3395 ©2004 by American Society of Hematology

The SSAO inhibitor BTT-2027 blocks inflammation in vivo. The SSAO inhibitor BTT-2027 blocks inflammation in vivo. Rats' air pouches were inflamed (carrageenan) or left untreated (saline), and the number of extravasated leukocytes was determined. The animals were treated with the indicated doses of SSAO inhibitors, vehicle (negative control), or dexamethasone (positive control). The numbers and subclasses of leukocytes lavaged from the air pouches are shown. The results are mean ± SEM (n = 8 in each group). The inhibitory effects of BTT-2027 on total leukocyte (P < .05) and on neutrophil (P < .05) extravasation were significant. Kaisa Koskinen et al. Blood 2004;103:3388-3395 ©2004 by American Society of Hematology

VAP-1 functions via 2 steps during PMN transmigration. VAP-1 functions via 2 steps during PMN transmigration. First, an antibody-dependent epitope is engaged to allow initial contacts between the PMNs and VAP-1. Then, an oxidative reaction takes place that ensures transmigration of the PMNs. If either step is blocked, the transmigration is halted. The PMN counter-receptor for the antibody-dependent epitope of VAP-1 and the leukocytic substrate for VAP-1 may be the same molecule or 2 separate molecules. Anti-VAP-1 mAbs do not interfere with entry of small model substrates or substrates expressed on the leukocyte surface into the enzymatically active site of VAP-1. Substr indicates substrate; Pretr, pretreated; and Transmigr, transmigration. Kaisa Koskinen et al. Blood 2004;103:3388-3395 ©2004 by American Society of Hematology