Lebectin, a Macrovipera lebetina Venom-Derived C-type Lectin, Inhibits Angiogenesis Both In Vitro and In Vivo ANTHONY PILORGET, 1,2 MAGALI CONESA, 1 SAMEH SARRAY, 3 JONATHAN MICHAUD-LEVESQUE, 2 SALMA DAOUD, 3 KWANG SIK KIM, 5 MICHEL DEMEULE, 2 JACQUES MARVALDI, 1 MOHAMED EL AYEB, 3 NAZIHA MARRAKCHI, 3,4 RICHARD BE ´ LIVEAU, 2 AND JOSE ´ LUIS 1 * 1 CNRS FRE 2737, Faculte´ de Pharmacie, bd Jean Moulin, Marseille, Cedex 5, France 2 Laboratoire de Me´decine Mole´culaire, Centre de Cance´rologie Charles-Bruneau, Hoˆpital Sainte-Justine-UQAM, Montreal, Quebec, Canada 3 Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Place Pasteur, Belve´de`re, Tunis, Tunisia 4 Faculte´ de Me´decine de Tunis, 9 Rue Professeur Zouheir Essafi, Tunisia 5 Division of Pediatric Infectious Diseases, Johns Hopkins University, School of Medicine, Baltimore, Maryland Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin–ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express avb3, avb5, and a5b1 integrins, as well as the a2, a3, a6, and b4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC 50  0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the a5b1 and aV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel TM (IC 50 ¼ 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel TM Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases. J. Cell. Physiol. 211: 307–315, 2007. ß 2007 Wiley-Liss, Inc. Angiogenesis is the formation of new capillaries from preexisting blood vessels (Folkman, 2004). The angiogenic process is fundamental to normal healing, reproduction and embryonic development (Zadeh and Guha, 2003). It has also been implicated in the pathogenesis of a wide variety of disorders, including primary and metastatic tumors, aneurysms, arteriovenous malformations, and cavernous malformations (Harrigan, 2003). The process of angiogenesis is complex, typically consisting of enzymatic degradation of the basement membrane, vascular endothelial cell migration into perivascular space, proliferation and alignment to form capillary-like structures, and new vessel formation (Folkman, 2001). Although growth factors stimulate new blood vessel growth and survival (Folkman, 1995b, 2004), adhesion to the extracellular matrix (ECM) also regulates endothelial cell survival, proliferation, and motility during new blood vessel formation (Li et al., 2003). At the molecular level, interactions between vascular cells and ECM are mainly mediated by integrins, a family of transmembrane proteins that link the ECM to the actin cytoskeleton within the cell (Hwang and Varner, 2004). Integrins are heterodimeric glycoproteins consisting of non-covalently associating a and b subunits. To date, 24 ab heterodimers, formed by combinations of 8 b and 18 a subunits, are known (Brakebusch et al., 2002). They are involved in various processes such as development, immune response, hemostasis, and maintenance of tissue integrity. Integrins also participate in various pathological processes such as chronic inflammation, tumor invasion, and metastasis (Hynes, 1992; Hemler, 1998). In addition to their role in cell adhesion, integrins relay molecular signals from the cellular environment which influence cell shape, survival, proliferation, Abbreviations: CLP, C-type lectin protein; HBMEC, human brain microvascular endothelial cell; ECM, extracellular matrix; CAM, chorioallantoic membrane. These two authors (Magali CONESA and Sameh SARRAY) equally contributed to this work. Contract grant sponsor: Association pour la Recherche sur le Cancer, France (ARC). Contract grant sponsor: Groupement des Entreprises Franc ¸aises dans la Lutte contre le Cancer (GEFLUC). Contract grant sponsor: Ligue Nationale contre le Cancer. Contract grant sponsor: Comite ´ Mixte franco-tunisien pour la Coope ´ration Universitaire (CMCU). Contract grant sponsor: National Sciences and Engineering Research Council of Canada (NSERC). These two authors (Magali CONESA and Sameh SARRAY) equally contributed to this work. *Correspondence to: Jose ´ Luis, CNRS FRE 2737, Faculte ´ de Pharmacie, 27, bd Jean Moulin, 13385 Marseille, Cedex 5, France. E-mail: jose.luis@pharmacie.univ-mrs.fr Received 31 July 2006; Accepted 5 October 2006 DOI: 10.1002/jcp.20935 ORIGINAL ARTICLE 307 ß 2007 WILEY-LISS, INC.