[CANCER RESEARCH 64, 8643– 8650, December 1, 2004] Elevated Flk1 (Vascular Endothelial Growth Factor Receptor 2) Signaling Mediates Enhanced Angiogenesis in  3 -Integrin–Deficient Mice Andrew R. Reynolds, 1 Louise E. Reynolds, 1 Tobi E. Nagel, 2 Julie C. Lively, 3 Stephen D. Robinson, 1 Daniel J. Hicklin, 4 Sarah C. Bodary, 5 and Kairbaan M. Hodivala-Dilke 1 1 Cell Adhesion and Disease Group, Tumour Biology Laboratory, Cancer Research UK Clinical Centre, Barts and the London, Queen Mary’s School of Medicine and Dentistry, John Vane Science Centre, London, United Kingdom; 2 Genentech, Inc., South San Francisco, California; 3 Center for Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; 4 ImClone Systems, Inc., New York, New York; and 5 DNAX, Palo Alto, California ABSTRACT Tumor growth, tumor angiogenesis, and vascular endothelial growth factor (VEGF)–specific angiogenesis are all enhanced in  3 -integrin–null mice. Furthermore, endothelial cells isolated from  3 -null mice show elevated levels of Flk1 (VEGF receptor 2) expression, suggesting that  3 -integrin can control the amplitude of VEGF responses by controlling Flk1 levels or activity. We now show that Flk1 signaling is required for the enhanced tumor growth and angiogenesis seen in  3 -null mice. Moreover,  3 -null endothelial cells exhibit enhanced migration and proliferation in response to VEGF in vitro, and this phenotype requires Flk1 signaling. Upon VEGF stimulation,  3 -null endothelial cells exhibit higher levels of phosphorylated Flk1 and extracellular-related kinases 1 and 2 than wild- type endothelial cells. Furthermore, signaling via ERK1/2 is required to mediate the elevated responses to VEGF observed in  3 -null endothelial cells and aortic rings in vitro. These data confirm that VEGF signaling via Flk1 is enhanced in  3 -integrin– deficient mice and suggests that this increase may mediate the enhanced angiogenesis and tumor growth ob- served in these mice in vivo. INTRODUCTION A favorable shift in the local concentrations of pro- and anti- angiogenic mediators is required for tumor neovascularization to occur (1). The expression of  v  3 and  v  5 integrin on endothelial cells has long been considered a pro-angiogenic event, because their expression is up-regulated on newly formed vessels (2, 3); whereas antagonists of  v  3 and  v  5 have been shown to inhibit pathological angiogenesis and tumor growth in animal models (2–5). Furthermore, the  3 -integrin antagonist, Vitaxin, is currently in clinical trials (6). However, we demonstrated recently that pathologic angiogenesis and tumor growth are actually enhanced in  3 -integrin– deficient or  3 /  5 -integrin doubly-deficient mice (7); these findings have brought into question the role of  3 -integrin as a positive regulator of angio- genesis (7, 8). Instead, we showed that the enhanced angiogenesis may be due to an elevated response to vascular endothelial growth factor (VEGF) in these mice. Because both VEGF and  3 -integrin signaling are prominent targets of anti-angiogenic therapy (9), the mechanism via which this enhanced response occurs requires addi- tional investigation. VEGF initiates tumor vascularization by driving an angiogenic response in endothelial cells (1, 10 –12). VEGF-A, the principal isoform of VEGF implicated in this process, mediates its effects via at least two receptor tyrosine kinases expressed on endothelial cells, VEGF receptor 1 (VEGFR1/Flt1) and VEGF receptor 2 (VEGFR2/ Flk1), and a coreceptor, neuropilin-1 (10, 12). Most reports agree that Flk1 activation is necessary and sufficient for angiogenic responses to VEGF-A (10, 12–14). However, Flt1 can have either a positive or negative effect on Flk1 signaling (12, 15–17), whereas neuropilin-1 can enhance signaling through Flk1, but can inhibit angiogenesis by binding to semaphorin ligands (18 –21). Tyrosine phosphorylation of Flk1 in response to VEGF activates numerous downstream signaling molecules, including the extracellu- lar-related kinases 1 and 2 (ERK1/2). ERK1/2 activity is a known mediator of endothelial cell proliferation, migration, and survival during angiogenesis (22–24). Furthermore, Flk1 expression is up- regulated on new blood vessels (25–27) and antagonists that block the function of VEGF-A or Flk1 inhibit tumor growth and angiogenesis in mice (9, 28 –32). Thus the level of Flk1 expression and the signaling activity of this receptor are considered to be of central importance to VEGF-specific angiogenesis. Endothelial cells isolated from  3 -null mice show elevated expres- sion levels of Flk1. We proposed that these raised levels of Flk1 expression might facilitate an enhanced response to VEGF in  3 -null endothelial cells (7), explaining the enhanced tumor and VEGF- specific angiogenesis observed in these mice. In the current report, we have used specific inhibitors of Flk1 to investigate this hypothesis in vivo and in vitro. We show that not only are the enhanced angiogen- esis and tumor growth observed in  3 -null mice dependent on VEGF binding to Flk1 in vivo, but that  3 -null endothelial cells show enhanced Flk1-dependent responses to VEGF in vitro. Furthermore, enhanced responses to VEGF in  3 -null endothelial cells requires ERK1/2 signaling. These results have important implications for the current understanding of how  v  3 integrin regulates VEGF signaling and tumor angiogenesis. MATERIALS AND METHODS Materials. All chemicals and reagents were from Sigma (Poole, United Kingdom), except for the following: DC101 rat monoclonal anti-Flk1 antibody and rat immunoglobulin G (IgG) control antibody (ImClone Systems, Inc., New York, NY), anti-ERK1, anti-ERK2, anti-HSC-70 (Autogen Bioclear, Oxfordshire, United Kingdom), anti-phospho-ERK1/2 (Cell Signaling Tech- nology, Hitchin, United Kingdom), anti-Fc III/II receptor, anti-Flk1, anti- CD31, anti-ICAM-2 (BD PharMingen, Oxford, United Kingdom), anti-Flk1- pY1173–specific antibody (Oncogene Research Products, San Diego, CA), anti-laminin-1 (Sigma), growth factor reduced–Matrigel (GFR-Matrigel; BD Biosciences, Oxford, United Kingdom), VEGF-A 164 (R&D Systems, Oxford, United Kingdom or Peprotech, London, United Kingdom), interferon- (IFN-; R&D Systems), endothelial cell growth mitogen (Biogenesis, Poole, United Kingdom), fetal calf serum (Biowest, Miami, FL), SU5416, UO126, fibronectin (Calbiochem, Beeston, United Kingdom), Vitrogen (ICN Bio- sciences, Irvine, CA), Ham’s F-12, and Dulbecco’s modified Eagle’s medium (DMEM; Cancer Research UK). Mouse Tumor Models. The B16F0 cell line used in these studies is a nonmetastatic variant of the B16 melanoma cell line (33). The CMT19T cell line is a mouse lung carcinoma cell line derived from the CMT167 cell line (34). Subcutaneous tumors were grown in age- and sex-matched wild-type or  3 -integrin– deficient mice on a mixed genetic background (C57BL6/129Sv). Two days before injection of tumor cells (day 0), wild-type and  3 -null mice received an intraperitoneal injection of DC101 antibody (800 g in 100 L of Received 8/2/04; revised 9/20/04; accepted 9/29/04. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Kairbaan M. Hodivala-Dilke, Cell Adhesion and Disease Group, Tumour Biology Laboratory, Cancer Research UK Clinical Centre, Barts and the London, Queen Mary’s School of Medicine and Dentistry, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, United Kingdom. Phone: 44-207-014-0406; Fax: 44-207-014- 0401; E-mail: Kairbaan.Hodivala-Dilke@cancer.org.uk. ©2004 American Association for Cancer Research. 8643 Research. on March 15, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from