Role of Vascular Endothelial Growth Factor in the Communication Between Human Osteoprogenitors and Endothelial Cells Maritie Grellier, 1,2 * Nancy Ferreira-Tojais, 1 Chantal Bourget, 1 Reine Bareille, 1 Fabien Guillemot, 1 and Joe ¨lle Ame ´de ´e 1 1 INSERM, U577, Bordeaux and Universite ´ Victor Segalen Bordeaux 2, UMR-S577, F-33076 Bordeaux Cedex, France 2 INEB—Instituto de Engenharia Biome ´dica, Divisa ˜o de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal ABSTRACT Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross-talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co-culture between osteoblastic and endothelial cells. Through a well defined direct contact co-culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular-like structures. VEGF 165 gene expression was detected in the HOPs, was up-regulated in the co-cultured HOPs and both Flt-1 and KDR gene expression increased in co- cultured HUVECs. However, the cell rearrangement observed in co-culture was promoted by a combination of soluble chemoattractive factors and not by VEGF 165 alone. Despite having no observable effect on endothelial cell tubular-like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co-culture-stimulated osteoblastic phenotype. This co- culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering. J. Cell. Biochem. 106: 390–398, 2009. ß 2009 Wiley-Liss, Inc. KEY WORDS: HUMAN STEM CELLS; ENDOTHELIAL CELLS; VEGF; CO-CULTURE; MIGRATION; OSTEOGENESIS B one remodeling consists of a balance of bone resorption and formation that occurs throughout life, not only in skeleton development but also in bone hemostasis and fracture healing. Bone formation is coordinated by osteoblasts and bone degradation is guaranteed by osteoclast activity. The formation and development of an active microvasculature is an essential stage for bone remodeling and fracture healing [Gerber and Ferrara, 2000; Carano and Filvaroff, 2003]. Angiogenesis, the formation of new blood vessels from pre-existing ones, allows for the restoration of blood flow to the fracture site and can modulate bone formation by the production of growth factors able to regulate osteoblastic activity, recruitment of stem cells and their orientation to the osteoblastic lineage [Fiedler et al., 2005]. Angiogenesis is modulated by a tight balance between pro- and anti-angiogenic factors and involves a cascade of events in which the migration of endothelial cells (ECs) is an initial event [Lamalice et al., 2007]. Chemotaxis refers to cell migration toward a gradient of soluble chemoattractants, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietins [Cross and Claesson-Welsh, 2001]. VEGF is a major promoter of both physiological and pathological angiogenesis that belongs to a family of homodimeric proteins consisting of six members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor [Cross et al., 2003; Ferrara et al., 2003]. VEGF-A exists in five different isoforms: VEGF 145 , VEGF 189 and VEGF 206 which are able to bind to the extracellular matrix (ECM) through heparin, VEGF 121 which is soluble and VEGF 165 , which is the most abundant form and can be both soluble and bound to the ECM [Neufeld et al., 1999]. These isoforms are able to activate two different tyrosine kinase receptors: VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) [Cross et al., 2003]. Their activation induces their phosphorylation and leads to the transduction of different signals promoting cell migration or osteoblastic phenotype Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 106:390–398 (2009) 390 Grant sponsor: ANR; Grant number: 06-PNANO-003. *Correspondence to: Maritie Grellier, 146 rue leo Saignat, Zone Nord, Bat 4A, Case 45, F-33076 Bordeaux Cedex, France. E-mail: maritie.grellier@inserm.fr Received 3 July 2008; Accepted 17 November 2008  DOI 10.1002/jcb.22018  2009 Wiley-Liss, Inc. Published online 6 January 2009 in Wiley InterScience (www.interscience.wiley.com).