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Several different approaches suggest that basement-membrane assembly is important for epithelial development. Basement membranes contain isoforms of collagen IV, proteoglycans, and noncollagenous glycoproteins such as the laminins and nidogens. The expression and role of laminins for epithelial morphogenesis is reviewed. Laminins are large heterotrimeric proteins composed of alpha, beta, and gamma chains. Many major epithelial laminins and their receptors have been identified recently, and the extracellular protein-protein interactions that drive basement-membrane assembly are beginning to be understood. Three laminin alpha-chains are typically made by epithelial, alpha 1, alpha 3, and alpha 5. Three major epithelial heterotrimers can at present be distinguished--laminin-1 (alpha 1 beta 1 gamma 1), laminin-5 (alpha 3 beta 3 gamma 2), and laminin-10 (alpha 5 beta 1 gamma 1)--but other heterotrimers may exist in epithelia. Laminins containing either alpha 1 or alpha 3 chains are largely limited to epithelia, whereas the alpha 5 is also found in endothelial and muscle basement membranes, particularly in the adult. Some epithelial cell types express several laminin alpha-chains, so it is relevant to test how the different laminins affect epithelial cells. Laminins interact with integrin type of receptors on the cell surface, but binding to other proteins has also recently been demonstrated. Two important recent discoveries are the identification of dystroglycan as a major laminin receptor in muscle and epithelia, and nidogen as a high-affinity laminin-binding protein important for basement-membrane assembly. Antibody perturbation experiments suggest that these protein-protein interactions are important for epithelial morphogenesis.

The sulfated glycosaminoglycan heparan sulfate (HS) is found ubiquitously on cell surfaces, in the extracellular matrix, and intracellularly as HS proteoglycans. Because of the structural heterogeneity of HS, tissue-derived HS preparations represent a mixture of HS chains originating from different cell types and tissue loci. Monoclonal anti-HS antibodies have been employed to detect the localization of specific HS epitopes in tissues, but limited information has been available on the saccharide structures recognized by the antibodies. We have studied the saccharide epitope structures of four anti-HS antibodies, HepSS1, JM13, JM403, and 10E4, which all recognize distinct HS species as demonstrated by different patterns of immunoreactivity upon staining of embryonic rat and adult human tissues. The epitopes recognized by JM13 and HepSS1 were found almost exclusively in basement membrane HS, whereas JM403 and 10E4 reacted also with cell-associated HS species. The binding of HepSS1, JM403, and 10E4 to HS was dependent on the GlcN N-substitution of the polysaccharide rather than O-sulfation. HepSS1 thus interacted with N-sulfated HS domains, JM403 binding was critically dependent on N-unsubstituted GlcN residues, and 10E4 bound to "mixed" HS domains containing both Nacetylated and N-sulfated disaccharide units. By contrast, JM13 binding seemed to require the presence of 2-O-sulfated glucuronic acid residues. Heparan sulfate (HS)1 proteoglycans on cell surfaces and in the extracellular matrix are implicated in developmental, regenerative, and pathological processes because of their interactions with multiple proteins (1-5). These interactions are mediated mainly via the HS components of the proteoglycans, which bind to growth factors/cytokines, matrix components, enzymes, and enzyme inhibitors and thereby regulate the tissue localization and biological activities of the proteins. Characterization of HS oligosaccharides with affinity to proteins such as antithrombin (6) and peptide growth factors (7) has led to identification of specialized protein binding HS domains with ligand-specific structural distinctions. These functional domains derive from enzymatic modification in the Golgi apparatus of the primary polymerization product of HS/heparin biosynthesis, composed of alternating glucuronic acid and Nacetylglucosamine units ((GlcUA-GlcNAc) n ). The nascent polymer is first subject to partial N-deacetylation/N-sulfation of the GlcNAc residues. The modification occurs in a regioselective fashion, giving rise to (i) consecutively N-sulfated regions, (ii) regions of alternating N-acetylated and N-sulfated disaccharide units, and (iii) domains that escape modification and remain N-acetylated (5, 8). Sometimes, the N-deacetylation/Nsulfation reaction remains partial, with N-unsubstituted GlcN units as a result (9, 10). The further modification reactions, C5 epimerization of GlcUA residues into iduronic acid (IdoUA) residues and O-sulfation, all occur in the vicinity of previously incorporated N-sulfate groups. O-Sulfation...

Leukocyte migration from the blood into tissues is pivotal in immune homeostasis and in inflammation. During the multistep extravasation cascade, endothelial selectins (P-and E-selectin) and vascular adhesion protein-1 (VAP-1), a cell-surfaceexpressed oxidase, are important in tethering and rolling. Here, we studied the signaling functions of the catalytic activity of VAP-1. Using human endothelial cells transfected with wild-type VAP-1 and an enzymatically inactive VAP-1 point mutant, we show that transcription and translation of E-and P-selectins are induced through the enzymatic activity of VAP-1. Moreover, use of VAP-1-deficient animals and VAP-1-deficient animals carrying the human VAP-1 as a transgene show a VAP-enzyme activity-dependent induction of P-selectin in vivo. Up-regulation of P-selectin was found both in high endothelial venules in lymphoid tissues and in flat-walled vessels in noninflamed tissues. VAP-1 activity in vivo led to in- IntroductionCoordinated function of the multistep leukocyte extravasation cascade is a prerequisite for leukocyte emigration from the blood into the tissue. Many adhesion and signaling molecules have well-established roles in this process. 1,2 On endothelial cells, selectins (P-selectin [CD62P] and E-selectin [CD62E]) mediate tethering of bloodborne cells to vascular endothelium, and the subsequent rolling along the endothelial lining in a sheardependent manner. 3 The rolling cells can be exposed to activating stimuli, such as chemokines, which can trigger firm, integrindependent adhesion of the leukocytes in the vessel. Finally, the leukocytes diapedese through the vessel wall using adhesion molecules from immunoglobulin and other superfamilies as well as local protease activity.In addition to these well-established interplayers, other molecules are involved in leukocyte trafficking. Among these, enzymes expressed on the cell surface that have their catalytic domains outside the plasma membrane (ectoenzymes) have emerging roles in leukocyte migration. 4 Vascular adhesion protein-1 (VAP-1, also known as amine oxidase copper containing-3 [AOC3]) is an ectoenzyme that belongs to the specific subgroup of oxidases known as semicarbazide-sensitive amine oxidases (SSAOs). 5,6 It catalyzes a reaction in which a primary amine is oxidatively deaminated into an aldehyde, and then hydrogen peroxide and ammonium are released. 7,8 VAP-1/SSAO is a bifunctional molecule that can support leukocyte adhesion under shear conditions via enzymeactivity-dependent and enzyme-activity-independent ways. 4 Monoclonal anti-VAP-1 antibodies that do not block its oxidase activity effectively block lymphocyte and granulocyte binding to endothelial cells in vitro and in vivo. Small-molecule SSAO enzyme inhibitors, on the other hand, are equally effective in perturbing leukocyte-endothelial contacts in vitro and in vivo. [9][10][11][12][13][14] The ability of the oxidase reaction to regulate the expression and/or function of other molecules involved in the emigration process is largely unknown.We h...