Antibody against Junctional Adhesion Molecule-C Inhibits Angiogenesis and Tumor Growth Chrystelle Lamagna, 1 Kairbaan M. Hodivala-Dilke, 2 Beat A. Imhof, 1 and Michel Aurrand-Lions 1 1 Department of Pathology and Immunology, Centre Me´dical Universitaire, Geneva, Switzerland and 2 Cell Adhesion and Disease Laboratory/Department of Tumour Biology, Bart’s and The London Queen Mary’s School of Medicine and Dentistry, John Vane Science Centre, London, United Kingdom Abstract The junctional adhesion molecule-C (JAM-C) was recently described as an adhesion molecule localized at interendothe- lial contacts and involved in leukocyte transendothelial mi- gration. The protein JAM-C interacts with polarity complex molecules and regulates the activity of the small GTPase Cdc42. The angiogenesis process involves rearrangement of endothelial junctions and implicates modulation of cell polarity. We tested whether JAM-C plays a role in angiogenesis using tumor grafts and hypoxia-induced retinal neovasculari- zation. Treatment with a monoclonal antibody directed against JAM-C reduces tumor growth and infiltration of macrophages into tumors. The antibody decreases angiogen- esis in the model of hypoxia-induced retinal neovasculariza- tion in vivo and vessel outgrowth from aortic rings in vitro . Importantly, the antibody does not induce pathologic side effects in vivo . These findings show for the first time a role for JAM-C in angiogenesis and define JAM-C as a valuable target for antitumor therapies. (Cancer Res 2005; 65(13): 5703-10) Introduction Angiogenesis is the formation of new blood vessels from the preexisting vasculature (1) and occurs in a multistep process involving migration, proliferation, and differentiation of endothelial cells leading to the formation of vascular loops (2). Angiogenesis occurs during embryonic development and adulthood during a variety of physiologic processes, including wound healing and the menstrual cycle of the endometrium (3, 4). Angiogenesis is also a key event in pathologic processes, such as tumor development (1) and diabetic retinopathy (5). The formation of immature endothelial sprouts is promoted by angiogenic factors, including vascular endothelial growth factor (VEGF) and angiopoietins. Maturation into functional vessels is then accomplished by the establishment of new interendothelial junctions, the organization of a new basement membrane, and the recruitment of pericytes (2). Altogether, these mechanisms result in the stabilization of a novel vascular network. Macrophages play an important role in regulating blood vessel formation by secreting angiogenic factors during tumor develop- ment as well as during physiologic angiogenesis (6, 7). Indeed, monocytes extravasate and migrate toward hypoxic or inflamma- tory regions created by a growing tumor (8). Although angiogen- esis is induced by angiogenic factors, additional molecules contribute to proliferation and remodeling of the vessel wall. As an example, VEGF leads to loosening of the pericyte-endothelial contacts, allowing proliferation and interaction of endothelial adhesion molecules with extracellular matrix (9). The integrins a v h 3 and a v h 5 participate in blood vessel development via a signaling cross-talk with receptors of angiogenic factors (10, 11). Other adhesion molecules implicated in the organization of interendothelial junctions are essential in maintaining integrity of the endothelium. For example, the targeted disruption in mice of the adherens junction molecule vascular endothelial-cadherin (VE-cadherin) leads to embryonic lethality due to impaired remodeling and maturation of vascular plexus (12, 13). More recently, it has been shown that the targeted disruption of the tight junction molecule, endothelial cell–selective adhesion mole- cule, inhibits angiogenesis in vitro and in vivo (14). In addition, in vitro experiments have shown that signaling through junc- tional adhesion molecule (JAM)-A and a v h 3 integrin is required for the angiogenic action of basic fibroblast growth factor (bFGF; refs. 15, 16). We recently described JAM-C and found it expressed in vascular cell-cell contacts (17, 18). When JAM-C is transfected into epithelial cells, it localizes in tight junctions, whereas it has been found in desmosomes of enterocytes (18, 19). We and others have shown that JAM-C is involved in leukocyte transendothelial migration (17, 18, 20, 21). Furthermore, JAM-C coimmunoprecipitates with polarity complex molecules, such as PAR-3, PAR-6, or PATJ, and regulates the activity of the small GTPase Cdc42 (22, 23). These results show that JAM-C plays a role in the formation and main- tenance of intercellular contacts and suggest that it may contribute to the remodeling of endothelial junctions. We therefore investigated whether JAM-C participates in angiogenesis, a mechanism involving rearrangement of endothelial junctions. Here, we show that a monoclonal antibody directed against JAM-C totally abolishes outgrowth of microvessels in ex vivo aortic ring assays. When injected in vivo , the antibody reduces hypoxia-induced angiogenesis in the retina and the growth of experimental tumors. These results show a role for JAM-C in angiogenesis and underline the importance of endothelial cell adhesion molecules in the formation of new blood vessels. Materials and Methods Antibodies. Rat monoclonal antibodies (CRAM) against human and mouse JAM-C (H33 for functional assays and H36 for immunocytochemistry) and rat monoclonal antibodies against mouse platelet/endothelial cell adhesion molecule-1 (PECAM-1)/CD31 (GC51) were described previously (18, 24). Anti-human CD44 (Hermes, 9B5) used as irrelevant antibody control rat IgG2a was kindly provided by Dr. B. Engelhardt (Theodor-Kocher- Institute, Bern, Switzerland) (25, 26). Monoclonal rat anti-mouse intercel- lular adhesion molecule-2 (ICAM-2; 3C4) and monoclonal rat anti-mouse Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Beat A. Imhof, Department of Pathology and Immunology, Centre Me ´dical Universitaire, 1 rue Michel-Servet, 1204 Geneva, Switzerland. Phone: 41-22-379-57-47; Fax: 41-22-379-57-46; E-mail: beat.imhof@medecine.unige.ch. 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