IntroductionWhite blood cells of the polymorphonuclear series (polymorphonuclear leukocytes [PMNs] or granulocytes) are practically absent from healthy tissues. Upon inflammation, however, rapid influx of PMNs from the blood into the affected tissue takes place. PMNs leave the blood mainly via postcapillary venules and they do not recirculate to any significant extent. Lymphocytes, on the other hand, not only exit at the sites of inflammation but also continuously recirculate between the blood and lymphoid tissues. The physiologic recirculation of lymphocytes is supported by specialized high endothelial venules in lymphoid tissues, and this leukocyte type returns back to the blood via the lymphatic vasculature. Monocytes, the third major leukocyte type, normally leave the blood at low levels in various tissues under normal conditions to replenish the tissue macrophage pools, and at high levels during the later phase of inflammation. The extravasation process of all leukocyte classes consists of a series of carefully controlled steps. [1][2][3] First, the leukocyte makes initial tethers with the luminal surface of the blood vessel and starts to roll along the endothelial lining. If it receives appropriate activation signals, it can adhere in a shear-resistant manner to the endothelium. Finally, the leukocyte transmigrates through the vascular wall and continues its odyssey toward the chemotactic inflammation-induced signals within the tissue. Multiple adhesion and signaling molecules act in concert to execute the emigration cascade. One of the endothelial molecules involved in lymphocyte trafficking is vascular adhesion protein-1 (VAP-1). 4 VAP-1 is a cell-surface enzyme belonging to a specific group of amine oxidases (semicarbazide-sensitive amine oxidases [SSAOs] Enzyme Commission 1.4.3.6) that catalyze oxidative deamination of primary amines. 5,6 Here we questioned what the possible mechanistic connection is between the adhesive and enzymatic activity of VAP-1. By using adenoviral constructs encoding native or mutated VAP-1, chemical inhibitors of SSAO activity, and function-blocking anti-VAP-1 monoclonal antibodies (mAbs) in in vitro flow chamber assays we were able to study the role of VAP-1 during PMN rolling, firm adhesion, and transmigration. Interestingly, VAP-1 primarily supported the transmigration step during the PMN extravasation process. Experiments with the SSAO inhibitors and VAP-1 mutants showed that the enzymatic activity of VAP-1 is a prerequisite for the adhesive function of this molecule. Finally, we showed in a rat model of acute inflammation that the new, specific, and potent SSAO inhibitor also blocks the recruitment of PMNs to affected tissue in vivo. These data show that VAP-1 functions in leukocyte adhesion in a step-wise fashion via separate antibody epitope-dependent and oxidation-dependent steps. These experiments are the first to show that an oxidative enzymatic reaction on the luminal surface of endothelial cells regulates PMN emigration in vitro and in vivo. Patients, materials, ...

The expression of human vascular adhesion protein-1 (hVAP-1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X-ray structure of hVAP-1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane-bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP-1, especially regarding the role of hVAP-1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N-glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent-exposed RGD motif at the entrance to each active site in hVAP-1 suggests that it may have a functional role.

We present a mechanism for agonist-promoted alpha(2A)-adrenergic receptor (alpha(2A)-AR) activation based on structural, pharmacological, and theoretical evidence of the interactions between phenethylamine ligands and alpha(2A)-AR. In this study, we have: 1) isolated enantiomerically pure phenethylamines that differ both in their chirality about the beta-carbon, and in the presence/absence of one or more hydroxyl groups: the beta-OH and the catecholic meta- and para-OH groups; 2) used [(3)H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [(3)H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition binding assays to determine binding affinities of these ligands to the high- and low-affinity forms of alpha(2A)-AR; 3) tested the ability of the ligands to promote receptor activation by measuring agonist-induced stimulation of [(35)S]GTPgammaS binding in isolated cell membranes; and 4) used automated docking methods and our alpha(2A)-AR model to predict the binding modes of the ligands inside the alpha(2A)-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlated the structural features of the ligands and ligand-receptor interactions with experimental ligand binding and receptor activation data. Based on the analysis, we show that structural rearrangements in transmembrane helix (TM) 5 could take place upon binding and subsequent activation of alpha(2A)-AR by phenethylamine agonists. We suggest that the following residues are important in phenethylamine interactions with alpha(2A)-AR: Asp113 (D(3.32)), Val114 (V(3.33)), and Thr118 (T(3.37)) in TM3; Ser200 (S(5.42)), Cys201 (C(5.43)), and Ser204 (S(5.46)) in TM5; Phe391 (F(6.52)) and Tyr394 (Y(6.55)) in TM6; and Phe411 (F(7.38)) and Phe412 (F(7.39)) in TM7.

Objective. Leukocyte traffic from the blood to the joints is crucial in the pathogenesis of arthritis. A bifunctional endothelial cell-surface glycoprotein, AOC3 (amine oxidase, copper-containing 3; also known as vascular adhesion protein 1), has both adhesive and enzymatic properties. We undertook this study to determine the contribution of AOC3 and its oxidase activity to leukocyte trafficking into inflamed joints in vivo.Methods. We used gene-modified animals, molecular modeling, an AOC3 enzyme inhibitor, oxidase assays, and arthritis models (adjuvant-induced arthritis [AIA] in rats and anti-type II collagen antibodyinduced arthritis in mice) to dissect the importance of AOC3 in vivo.Results. The AOC3 inhibitor fitted well with a covalent binding mode into the active site of the AOC3 crystal structure. It selectively blocked the oxidase activity of AOC3 in enzyme assays. Intraperitoneal and oral administration of the AOC3 inhibitor significantly ameliorated rat AIA. In anti-type II collagen antibodyinduced arthritis in mice, the AOC3 inhibitor also improved the outcome of the joint inflammation. The acute semicarbazide-sensitive amine oxidase blockade by the inhibitor had even more pronounced effects than genetic deletion of AOC3. Enzymatic analyses showed that the inhibitor also blocked 2 other structurally very closely related AOCs, but not any of more than 100 other enzymes tested.Conclusion. These are the first data to demonstrate that the enzymatic activity of the atypical endothelial adhesion molecule AOC3, and possibly that of other closely related ecto-oxidases, is crucial for leukocyte exit from the vessels in inflamed joints in vivo.