**1. Introduction**

152 Hepatocellular Carcinoma – Basic Research

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The extravasation of leukocytes from the vasculature to the tissue space is a fundamental response of the normal immune system. A multistep model of leukocyte adhesion to vascular endothelium has been characterized, although details of the signaling differ between tissues 1-9.

Lymphocytes recirculate between blood and tissues as part of the immune surveillance process, and recent evidence suggests that specialized subsets of T cells exhibit discrete tissue-specific patterns of recirculation in vivo 2, 10.

These cells are directed to particular tissues by combinations of adhesion molecules and chemokines that control the lymphocyte recognition of and adhesion to endothelium. For example, memory T cells that recirculate to the gut lamina propria express integrin α4β7 and bind to an endothelial ligand, namely, mucosal cell adhesion molecule-1, which is detected in gut endothelium 11. Conversely, T cells that migrate to the skin do not express α4β7 but do express high levels of the lymphocyte Ag, Which binds to E-selectin on dermal endothelium 12 .The liver is a major site of Ag exposure and contains large numbers of lymphocytes even under normal conditions. These lymphocytes are differentiated lymphocytes that are displaced by apoptosis 13-17. It is thus likely that tissue-specific signals regulate lymphocyte recruitment to the liver 18.

The hepatic cells are formed into ranks by specialized endothelium that supports lymphocyte adhesion and recruitment in a low-shear environment 19. Hepatic endothelium has a discrete phenotype compared with endothelium from other vascular beds.

Several cell adhesion molecules play an important role in this complex process and stabilize the adhesion and diapedesis of leukocytes across the endothelial barrier, similar to the manner in which human hepatic endothelium cells secrete the endothelial adhesion molecule vascular adhesion protein-1 (VAP-1)-9.

<sup>\*</sup> Corresponding Author

Vascular Adhesion Protein-1 and Hepatocellular Cancer 155

exposed to activating stimuli such as chemokines that can reinforce the integrin-dependent adhesion of leukocytes and migration through vessel walls using adhesion molecules from immunoglobulin and other super-families as well as local protease activity. Immune function is predicated on the continuous exchange of lymphocytes between the blood and tissue and is regulated by molecular interactions between the circulating lymphocytes and

VAP-1 was described after monoclonal antibody (mAB) 1B2 immunoprecipitation, which gave a 170- to 180-kDa homodimeric sialoglycoprotein that was formed by two 90 kDa subunits, bound by disulfide bonds, with close sequence homology to the copper-dependent semicarbazide-sensitive amine oxidases (SSAO) 1, 20. Both the transmembrane and soluble forms of VAP-1 exhibit monoamine oxidase activity 29. A rabbit homolog of VAP-1 has been shown to function under in vivo conditions during the primary temporary interactions between endothelial and lymphoid cells 20. VAP-1 has a large extracellular domain, a single-

Soluble adhesion molecules may have definite and adverse physiological effects. They may function as inhibitors of cell to cell adhesion by competing with their membrane-bound

Serum measurements of VAP-1 were performed by enzyme-linked immunosorbent assay (ELISA). Blood was drawn into serum tubes and allowed to clot for at least 30 min, centrifuged at 3000 rpm for 10 min and kept frozen at -70°C 22. Wells of microtiter plates were coated with 100 µl of the anti–VAP-1 mAb TK8-18 at 10 µg/ml in 0.1 M NaHCO3 buffer (pH 9.6), stored at 40Covernight, and then kept at 37°C for 1 h. The wells were washed 6 times with 0.1% Tween 20 in phosphate buffered saline (PBS) and then blocked by the addition of 200 µl of PBS containing 1% gelatin and 1% nonfat milk powder (blocking solution) for 45 min at room temperature to prevent nonspecific adsorption. After washing the wells 6 times with Tween/PBS, 175 µl of each serum sample was added to the wells, and the plates were left at room temperature for 1 h. The wells were then washed six times with Tween/PBS and incubated with 100 µl of the biotinylated anti–VAP-1 mAbTK8-14 or biotinylated control mAb Hermes-3 at room temperature for 1 h. After six washes with Tween/PBS, 100 µl of streptavidin–horseradish peroxidase was added to the wells, and the plates were allowed to incubate at room temperature again for 1 h. Thereafter, the plates were washed six times with Tween/PBS and finally developed with a chemoluminescence ELISA reagent (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions. All serum samples were measured at 1:25 dilution. Each sample was measured in triplicate, and the anti–VAP-1 value was calculated by subtracting

the mean background value of the negative control from the mean value of VAP-1 23.

standard curve of titrated tonsil lysate using a linear regression analysis 23.

Each assay included a titration of the quantified tonsil lysate; this titration was used to generate a standard curve. To obtain a protein milieu similar to the serum samples, tonsil lysate standards were diluted in blocking solution that contained as much VAP-1–depleted human serum as the test samples contained patient serum. Calculations of the amount of sVAP-1 in the serum samples were made by comparing the specific VAP-1 values with a

The adhesive function of VAP-1 is important in optimal oligosaccharide modifications since desialylated VAP-1 does not bind to lymphocytes 30. The lymphocyte ligand of VAP-1 is

ligands on the surface of endothelial cells 28.

forms.

pass transmembrane domain, and a short cytoplasmic cauda 20.

The VAP-1 molecule is a 170 kDa homodimeric glycoprotein that consists of two 90 kDa subunits that are held together by disulfide bonds 20. VAP-1 has a large extracellular domain, a single-pass transmembrane domain, and a short cytoplasmic cue 21. The molecule has ample sialic acid moieties that are required for its adhesive function, which was shown based on the inability of VAP-1 to mediate lymphocyte adhesion to desialylated vessels 20 . The leukocyte ligand for VAP-1 is presently unknown.

Serum measurements of VAP-1 are performed by enzyme-linked immunosorbent assay22.

A possible role for VAP-1 was shown in adhesion assays. VAP-1 may have a normal physiological role in maintaining the proadhesive status of PBLs toward endothelia since its concentration in normal individuals is within the range that is used in these adhesion experiments.

Some reports have shown that significantly elevated levels of VAP-1 were found in patients with liver diseases 22, 23. Patients had increased systemic circulation, and VAP-1 levels in these patients were higher, suggesting that VAP-1 could engender biological effects. It is interesting that patients with hepatocellular carcinoma had higher VAP-1 values than patients with inflammatory liver diseases, which agrees well with the finding of high VAP-1 expression in primary liver tumors but not in liver metastases 22-24. Other non-hepatic inflammatory diseases were not related to increased levels of VAP-1 23. An increase in VAP-1 levels is specific to particular inflammatory diseases. The specificity of increased VAP-1 levels for certain liver diseases (hepatocellular carcinoma) justifies further characterization of the role of VAP-1 in the inflammatory activity of these diseases.

In conclusion, VAP-1 is present in the serum of healthy individuals at a level similar to those of most other soluble endothelial adhesion molecules 25. The concentration of VAP-1 is higher in certain liver diseases than the levels of other known circulating endothelial adhesion molecules 23, 25.
