VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

ARTICLE Received 27 Jul 2012 | Accepted 28 Feb 2013 | Published 9 Apr 2013 DOI: 10.1038/ncomms2683 OPEN VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation Makoto Hayashi1, Arindam Majumdar1,2, Xiujuan Li1, Jeremy Adler1, Zuyue Sun1, Simona Vertuani2, Carina Hellberg3, Sofie Mellberg1, Sina Koch1, Anna Dimberg1, Gou Young Koh4, Elisabetta Dejana5, Heinz-Georg Belting6, Markus Affolter6, Gavin Thurston7, Lars Holmgren2, Dietmar Vestweber8 & Lena Claesson-Welsh1 Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp  /  teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation. 1 Uppsala University, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjölds v. 20, 751 85 Uppsala, Sweden. of Oncology and Pathology, Cancer Center Karolinska (CCK), Karolinska Institutet, 171 76 Stockholm, Sweden. 3 School of Biosciences, University of Birmingham, B15 2 TT Birmingham, UK. 4 Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 305-701 Korea. 5 IFOM-IEO Campus Via Adamello, 16-20139 Milan, Italy. 6 Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. 7 Regeneron Pharmaceuticals Inc, 777 Old Saw Mill River Road, Tarrytown, New York 10591, USA. 8 Max Planck Institute for Molecular Biomedicine, Röntgenstrae 20, 48149 Münster, Germany. Correspondence and requests for materials should be addressed to L.C.-W. (email: ). 2 Department NATURE COMMUNICATIONS | 4:1672 | DOI: 10.1038/ncomms2683 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2683 V ascular endothelial growth factor (VEGF)-A (henceforth, denoted as VEGF) is essential for blood vessel development during embryogenesis, for angiogenesis in the adult and for regulation of vascular permeability1. VEGF binds to two receptor tyrosine kinases, VEGFR1 and VEGFR2. Whereas VEGFR1 primarily serves a negative regulatory role, VEGFR2 transduces all known effects of VEGF2. Gene targeting of vegfa and vegfr2 both result in early embryonic lethality due to arrested endothelial cell (EC) differentiation3–5. Binding of VEGF to VEGFR2 induces receptor dimerization, activation of the kinase and autophosphorylation of tyrosine residues6,7. Autophosphorylated residues regulate kinase activity and bind signal transducers that propagate signals eventually resulting in EC survival, proliferation, migration and lumen formation. Kinase activity is tightly regulated, for example, through protein tyrosine phosphatases (PTPs). Vascular endothelial (VE) protein tyrosine phosphatase (VE-PTP in the mouse; PTP-receptor beta; PTP-RB in the human) is specifically expressed in ECs8,9. Inactivation of the ve-ptp gene results in normal vasculogenesis, but abnormal angiogenesis and failure to organize the vasculature into higher-order branched vessels, leading to embryonic death at E11 (refs 8,9). VE-PTP dephosphorylates substrates at EC junctions, such as the receptor tyrosine kinase Tie2 (ref. 10), and adherens junction components VE-cadherin11 and its partner plakoglobin12. Tie2, and its activating ligand Angiopoietin-1 (Ang1) are required for vessel integrity13. Ang1 promotes formation of Tie2/VE-PTP complexes at cell–cell contacts, thereby regulating junctional stability14. Phosphorylation of VE-cadherin is accompanied by loosening of adherens junctions and vascular permeability. VEcadherin silencing or gene targeting in vitro15, in zebrafish16 and in mice17 disturb endothelial polarization and vessel lumen formation. Interactions between VE-cadherin and PAR3 and PAR6, members of the cell polarity complex18,19, may be critical for the polarization and lumen formation processes. We have previously shown that VE-PTP associates with and dephosphorylates VEGFR2 (ref. 20). We now show that VEPTP regulates junctional activity of VEGFR2 in a Tie2-dependent manner, accompanied by decreased VE-cadherin phosphorylation. Overall, VE-PTP serves as a molecular relay to orchestrate junctional maturation, EC polarization and lumen formation during VEGF-driven sprouting angiogenesis. Results Angiogenic sprouting in VE-PTP-deficient embryoid bodies. We analysed sprouting angiogenesis in mouse embryoid bodies (EBs) from wild-type (WT) and ve-ptp  /  embryonic stem cells (ESCs)8,21. Ve-ptp  /  EBs formed a denser network of vessel sprouts with similar length but with increased area compared with WT EBs (Fig. 1a–c). There was a tenfold increase in CD31/ VE-cadherin double-positive ECs in VEGF-treated ve-ptp  /  EBs compared with VEGF-treated wild-type EBs (Fig. 1d). Moreover, the ve-ptp  /  ECs extended numerous long filopodia throughout the sprout, while most WT stalk cells did not (Fig. 1e). We hypothesized that increased EC proliferation and filopodia formation might be due to elevated VEGFR2 activity. Indeed, immunostaining for VEGFR2 and the VEGFR2 phosphorylation site pY1175 (Fig. 1f) showed increased levels in ve-ptp  /  sprouts compared with WT (Fig. 1g). The pVEGFR2/total VEGFR2 ratio was significantly higher in the ve-ptp  /  stalks compared with WT stalks. The pVEGFR2 activity often colocalized with CD31 immunostaining, which was used to identify EC junctions (Fig. 1h). The VEGFR2 and pVEGFR2 stainings did not always colocalize, possibly because 2 the antibodies against pVEGFR2 and VEGFR2 detected receptor intra- and extracellular domains, respectively. Immunostaining for VE-PTP also showed junctional localization (Fig. 1i). Supplementary Figure S1a,b shows that VE-PTP ablation was accompanied by reduced pericyte coating, indicating immature sprouts. Moreover, whereas ve-ptp transcripts were efficiently eliminated after gene targeting, there was no change in expression levels of genes known to affect angiogenic sprouting, s (...truncated)