993 RESEARCH ARTICLE INTRODUCTION Vasculogenesis and angiogenesis are the main processes responsible for embryonic vascular development. The formation of new vascular tubes comprises endothelial cell proliferation and migration as well as the inhibition of apoptosis. Moreover, the surrounding extracellular matrix (ECM) undergoes degradation and remodeling during vascular sprouting. The molecules involved in ECM-cell signaling are the integrin receptors, which are characterized by their bidirectional (outside-in and inside-out) signaling. Integrins bind to ECM proteins such as collagen, laminin and fibronectin and thereby regulate stabilization, migration and the morphology of vascular structures (Senger et al., 2002; Ruoslahti and Engvall, 1997). Furthermore, integrins interact with intracellular cytoskeletal proteins, providing structure and morphology to the cells (Pozzi and Zent, 2003). Thus, the role of integrins in matrix adhesion and cytoskeletal reorganization is crucial for cell growth, survival and migration. These key processes are regulated via several integrin- dependent cross-linked intracellular pathways involving AKT and NO (Abair et al., 2008; Basile et al., 2007; Viji et al., 2009). Moreover, there is also evidence that integrins are involved in the development of vessels, as deletion of the a5 chain reduced capillary plexus formation (Francis et al., 2002) and avb3 integrins were found to be upregulated in angiogenesis (Brooks et al., 1994). In particular, b1 integrins have pleiotropic and important functions in vascular development as they were shown to be necessary for teratoma growth and vasculogenesis in embryoid bodies (EBs) (Bloch et al., 1997). These data are supported by the endothelial- specific deletion of b1 integrins causing early embryonic lethality of mice due to vascular defects (Carlson et al., 2008; Lei et al., 2008; Tanjore et al., 2008). To investigate the cellular mechanisms underlying these abnormalities, we chose the embryonic stem (ES) cell system, which is an excellent model to examine the cell biological effects of mutations resulting in early embryonic lethality. We found that b1 integrins regulate key steps of vascular development and network formation, such as endothelial cell maturation, migration and elongation. Proliferation and apoptosis were enhanced by activation of the signaling molecules AKT (AKT1 – Mouse Genome Informatics) and eNOS (NOS3), respectively. Thus, our study provides novel mechanistic insight into the role of b1 integrins for neo-angiogenesis/vascularization. MATERIALS AND METHODS Cell culture The mouse blastocyst-derived ES cell lines D3 (wild-type) and b1 integrin –/– were maintained in culture as described by Fassler (Fassler et al., 1996). For EB formation cells were cultured in hanging drops for 2 days. Subsequently, the aggregates were incubated for 3 days in suspension and plated on day 5 in a 24-well plate on gelatine-coated coverslips. Experiments were performed after 7, 12, 14 and 18 days, referred to as 5+7, 5+12, 5+14 and 5+18. Vital microscopy or time-lapse microscopy In order to live monitor early stages of vasculogenesis, ES cells of the D3 or b1 integrin –/– line were transfected with a PECAM/EGFP construct; after differentiation, clusters of ES cell-derived endothelial precursors were continuously observed for up to 10 hours (Kazemi et al., 2002) with an inverted microscope (Axiovert 100, Zeiss, Goettingen, Germany) equipped Development 137, 993-1002 (2010) doi:10.1242/dev.045377 © 2010. Published by The Company of Biologists Ltd 1 Institute of Physiology I, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany. 2 German Sport University, Department of Molecular and Cellular Sport Medicine, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany. 3 Department of Urology, University Medical Center of Cologne, 50924 Cologne, Germany. *These authors contributed equally to this work † Authors for correspondence (bernd.fleischmann@uni-bonn.de; w.bloch@dshs- koeln.de) Accepted 20 January 2010 SUMMARY b1 integrins are important regulators of vascular differentiation and development, as their endothelial-specific deletion results in embryonic lethality. In the present study, we investigated the molecular mechanisms underlying the prominent vascular abnormalities that occur in the absence of b1 integrins. Because of the early embryonic lethality of knockout mice, we studied endothelial cell and vessel development in b1-integrin-deficient murine embryonic stem cells to gain novel insights into the role of b1 integrins in vasculo-angiogenesis. We found that vessel development was strongly defective in the mutant embryoid bodies (EBs), as only primitive and short sprouts developed from clusters of vascular precursors in b1 integrin –/– EBs, whereas complex network formation of endothelial tubes was observed in wild-type EBs. The vascular defect was due to deficient b1 integrin expression in endothelial cells, as its endothelial-specific re-expression rescued the phenotype entirely. The mechanism responsible for defective vessel formation was found to be reduced endothelial cell maturation, migration and elongation. Moreover, the lower number of endothelial cells in b1 integrin –/– EBs was due to an increased apoptosis versus proliferation rate. The enhanced apoptosis and proliferation of b1 integrin –/– endothelial cells was related to the elevation of peNOS and pAKT signaling molecules, respectively. Our data demonstrate that endothelial b1 integrins are determinants of vessel formation and that this effect is mediated via different signaling pathways. KEY WORDS: b1 integrin, Embryonic stem cells, Vasculogenesis, Basement membrane, Mouse Endothelial b1 integrins regulate sprouting and network formation during vascular development Daniela Malan 1, *, Daniela Wenzel 1, *, Annette Schmidt 2 , Caroline Geisen 1 , Axel Raible 3 , Birgit Bölck 2 , Bernd K. Fleischmann 1,† and Wilhelm Bloch 2,† DEVELOPMENT