However, using live cell staining and confocal microscopy, we observed that the MAdCAM-1 protein was redistributed onto

the surface of HECs stimulated with TNF-α and MA (Fig. 1B). In addition, we found that MAdCAM-1 was released in a soluble form (sMAdCAM-1) in the supernatant of TNF-α–treated and MA-treated HECs in comparison with media alone (Fig. 1C). Therefore, we have shown that MA and TNF-α up-regulate MAdCAM-1 mRNA expression in HECs, induce protein redistribution onto the cell surface, JNK inhibitor and promote increased secretion of sMAdCAM-1. To study the function of HEC-expressed MAdCAM-1, we used flow-based adhesion assays with JY cells, which express high levels of the MAdCAM-1 receptor α4β7 on the cell surface (Fig. 2A). JY cells were perfused over HEC monolayers at 0.05 Pa, and adhesion was recorded. Under basal conditions, no adhesion was detected; however, stimulation of HECs with TNF-α and MA significantly increased the total number of adherent cells, and this was reduced by an antibody blockade of MAdCAM-1 (P1) or α4β7 (ACT-1; Fig. 2B). The IMC antibody GSK-3 cancer that was used showed no inhibitory effect [109 ± 21 adherent

cells/mm2/106 perfused cells (standard error of the mean) in HECs treated with TNF-α and MA and 116 ± 41 adherent cells/mm2/106 perfused cells in TNF-α and MA and isotype control stimulated HEC]. Altogether, our data show that TNF-α and MA induce the redistribution of the MAdCAM-1 protein onto the cell surface and render it functionally active to support the binding of α4β7+ JY cells. To validate the role of VAP-1/SSAO in MAdCAM-1 induction, we used adenoviral constructs encoding enzymatically active and inactive hVAP-1. The enzyme activities of the constructs were confirmed before use (Supporting

Information Fig. 2). More than 95% of HECs transfected with the adenoviral constructs expressed hVAP-1 on their surface (Fig. 3A), with similar median channel fluorescence values for the two constructs (197 ± 40 for hVAP-1 and 216 ± 40 for hVAP-1_Y471F, n = 7 Linifanib (ABT-869) HECs). We then exposed transfected HECs to MA and TNF-α and observed increased MAdCAM-1 protein levels in HECs transfected with enzymatically active hVAP-1 (Fig. 3B1). Under control conditions, the presence of WT hVAP-1 caused a significant increase in comparison with HECs transfected with the mutant hVAP-1, probably as a result of endogenous ligands. When HECs were stimulated with TNF-α and MA in the presence of WT hVAP-1, there was a significant increase in MAdCAM-1 expression in comparison with HECs transfected with mutant hVAP-1 (Fig. 3B2). To further confirm the role of VAP-1/SSAO in MAdCAM-1 induction, we studied the effects of the end products released by MA deamination by VAP-1.