Research paper Proliferation and migration activities of fibroblast growth factor-2 in endothelial cells are modulated by its direct interaction with heparin affin regulatory peptide C  elia Dos Santos a, *, 1 , Charly Blanc b , Rania Elahouel a , Mark Prescott c , Gilles Carpentier a , Alessandro Ori c, 2 , Jos  e Courty a , Yamina Hamma-Kourbali a, 3 , David G. Fernig c , Jean Delb  e a, 3 a Laboratoire CRRET, CNRS, Universit e Paris Est, Avenue du G en eral de Gaulle, 94010 Cr eteil Cedex, France b IMRB INSERM, U955, Equipe 07, Facult e de M edecine, 8 rue du G en eral Sarrail, 94010 Cr eteil, France c Department of Structural and Chemical Biology, Institute of Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK article info Article history: Received 3 April 2014 Accepted 2 October 2014 Available online xxx Keywords: Growth factors Interaction Proliferation Migration Protect and label abstract Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. In normal or pathological angiogenesis, angiogenic growth factors activate cognate receptors on endothelial cells. Fibroblast growth factor-2 (FGF-2) and heparin affin regulatory peptide (HARP) are two heparin-binding growth factors and were described for their pro-angiogenic properties on human umbilical endothelial cells (HUVEC). We now show that HARP acts as a mediator of FGF-2's stimulatory effects, since it is able to inhibit the proliferation and migration of HUVEC induced by FGF-2. We demonstrate by ELISA and optical biosensor binding assay that HARP and FGF-2 interact through direct binding. We have adapted a previously developed structural proteomics method for the identification of residues involved in proteineprotein interactions. Application of this method showed that two se- quences in HARP were involved in binding FGF-2. One was in the C-thrombospondin type 1 repeat (C- TSR-1) domain and the other in the C-terminal domain of HARP. The identification of these regions as mediating the binding of FGF-2 was confirmed by ELISA using synthetic peptides, which are as well mediators of FGF-2-induced proliferation, migration and tubes formation on HUVEC in vitro. These re- sults imply that besides a regulation of the proliferation, migration and angiogenesis of HUVEC by direct interaction of FGF-2 with its receptors, an alternative pathway exists involving its binding to growth factors such as HARP. © 2014 Elsevier B.V. and Soci et e française de biochimie et biologie Mol eculaire (SFBBM). All rights reserved. 1. Introduction Angiogenesis plays a key role in physiological and physiopath- ological processes. The formation of new capillaries from the endothelium of existing vasculature involves different steps, including degradation of the basement membrane, migration and proliferation of endothelial cells and stabilization of the new vascular tubes [1]. This process is tightly regulated by the balance between different pro and anti-angiogenic molecules, including growth factors. Among the pro-angiogenic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) represent the most important and extensively studied. Abbreviations: a.a., amino acid; ALK, anaplastic lymphoma kinase; ASP, aspartic acid; CTGF, connective tissue growth factor; FGF-2, fibroblast growth factor-2; GLU, glutamic acid; HARP, heparin affin regulatory peptide; HBGF, heparin binding growth factors; HS, heparan sulfates; HUVEC, human umbilical vein endothelial cells; MK, midkine; RPTP b/z, receptor-type protein tyrosine phosphatase beta/zeta; VEGF 165 , vascular endothelial growth factor 165; TSR-1, thrombospondin type 1 repeat. * Corresponding author. Tel.: þ33 145 17 14 47; fax: þ33 145 17 18 16. E-mail address: ccliadossantos@gmail.com (C. Dos Santos). 1 C elia Dos Santos was funded by a Marie Curie Intra-European fellowship. This work was supported in part bya grant from ANR (ANR-06-RIB 016-02), a grant from INCA (PL06-093) and by the Centre National de la Recherche Scientifique. 2 Alessandro Ori's present address is Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg 69117, Germany. 3 Jean Delb e’s, and Yamina Hamma Kourbali's present address is IMRB INSERM, U955, Equipe 07, Facult e de M edecine, 8 rue du G en eral Sarrail, 94010 Cr eteil, France. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi http://dx.doi.org/10.1016/j.biochi.2014.10.002 0300-9084/© 2014 Elsevier B.V. and Soci et e française de biochimie et biologie Mol eculaire (SFBBM). All rights reserved. Biochimie xxx (2014) 1e8 Please cite this article in press as: C. Dos Santos, et al., Proliferation and migration activities of fibroblast growth factor-2 in endothelial cells are modulated by its direct interaction with heparin affin regulatory peptide, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.10.002