The CXC-Chemokine CXCL4 Interacts with Integrins Implicated in Angiogenesis Sallouha Aidoudi1,2*, Kinga Bujakowska1,2, Nelly Kieffer3, Andreas Bikfalvi1,2* 1 INSERM, U920, Talence, France, 2 Univ Bordeaux, Talence, France, 3 Laboratoire de Biologie et Physiologie Inte´gre´e (CNRS/GDRE-ITI), University of Luxembourg, Luxembourg City, Luxembourg Abstract The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet a-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with avb3 on the surface of avb3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through avb3 integrin, and also through other integrins, such as avb5 and a5b1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect. Citation: Aidoudi S, Bujakowska K, Kieffer N, Bikfalvi A (2008) The CXC-Chemokine CXCL4 Interacts with Integrins Implicated in Angiogenesis. PLoS ONE 3(7): e2657. doi:10.1371/journal.pone.0002657 Editor: Thomas Zwaka, Baylor College of Medicine, United States of America Received March 8, 2008; Accepted June 10, 2008; Published July 16, 2008 Copyright: 2008 Aidoudi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by grants from the ‘‘Agence Nationale de la Recherche (ANR)’’, the National French Cancer Institute (INCA), ‘‘the Ligue Nationale du Cancer’’ and the ‘‘Association pour la Recherche sur le Cancer (ARC)’’ to A.B. The sponsors had no role in designing the study or approving the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: salouaaidoudi@yahoo.fr (SA); a.bikfalvi@angio.u-bordeaux1.fr (AB) Introduction Angiogenesis is the formation of new capillaries from preexisting blood vessels. Angiogenesis plays an important role in physiologic processes such as wound healing and in disease progression such as cancer, diabetic retinopathy and various inflammatory disorders [1]. In particular, the expansion of solid tumors and other cancers critically depends on angiogenesis [2], making anti-angiogenesis strategies relevant for cancer therapy [3]. CXCL4, a CXC- chemokine, is synthesized predominantly in megakaryocytes, sequestered in a-granules of platelets, and released upon activation of platelets [4]. CXCL4 and the peptide derived from its carboxyl- terminal domain (CXCL4/CTF) display a strong antiangiogenic activity in vitro [5–7] and in vivo [5,7–8]. They suppress growth of various tumors [9–11] and metastasis [12] in vivo. This effect is related to their antiangiogenic action and not to tumor cell proliferation [8–12]. Although CXCL4 is one of the first agents discovered to have an antiangiogenic action in ex-vivo systems [5], the specific receptor mechanisms that transduce the antiangio- genic signal of CXCL4 are still poorly understood. Angiogenesis depends on vascular endothelial cell proliferation, migration and invasion. A family of adhesion receptors known as integrin receptors tightly regulates these early angiogenic processes. Indeed, integrins are the major adhesion receptors used by endothelial cells undergoing angiogenesis to interact with their extracellular matrix (ECM). This interaction causes spreading of endothelial cells with cytoskeleton re-organization events necessary for cells to invade ECM, to proliferate, to migrate and to ultimately form new tubular vessels [13]. The integrin-dependency of tumor angiogenesis in vivo is evidenced by the fact that antagonists of the avb3 integrin, which are highly expressed in angiogenic endothe- lium, suppress tumor growth by inhibiting angiogenesis [14,15]. Furthermore, the functionally and structurally homologous avb5 has been implicated in angiogenesis under certain conditions and selective antagonists of avb5 or dual antagonists of avb3 and avb5 integrins inhibit VEGF-stimulated angiogenesis in vivo in animal models [16]. Finally, the a5b1 integrin was shown to play a crucial role in angiogenesis and selective antagonists of a5b1 integrin block tumor angiogenesis, thereby causing regression of human tumors in animal models [17]. Several integrin inhibitors are currently tested as therapeutics for cancer [3]. Taking into account that CXCL4 is released from the a- granules of activated platelets in the vicinity of vessel wall injury [18] and that CXCL4 targets the endothelial cells in vivo that undergo active angiogenesis [19,20], we examined the possibility that CXCL4 might function as a ligand for integrins. We show here that CXCL4 binds to avb3 and to some extent to avb5 and a5b1 integrins on the surface of endothelial cells. The CXCL4- integrin interaction is of functional significance, since CXCL4 modulated endothelial cell functions, such as spreading and migration through integrins. Taken together with the established importance of integrin in tumor angiogenesis, this study provides a new mechanistic context for the function of CXCL4 as an angiogenesis inhibitor. PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2657