fms-like tyrosine kinase (FlT)-1 and VeGFr2/Kdr/ fetal liver kinase (FlK)-1. experimental evidence supports VeGFr2 as the major receptor mediating cell signaling in endothelial cells in response to VeGF. Selective activation of VeGFr1 does not induce cell migration or intercel-

nitrogen-containing bisphosphonates (BiS) are potent inhibitors of bone resorption and are used in the treatment of a number of medical conditions, including multiple myeloma, breast cancer and osteoporosis. recent experimental evidence demonstrates that BiS also affect endothelial cell functions and angiogenesis; however, the molecular mechanism(s) are unclear. Vascular endothelial growth factor (VeGF) is a potent pro-angiogenic signal for endothelial cells. BiS inhibit VeGF responses in endothelial cells. The VeGF receptor-2 (VeGFr2) is the main signaling receptor for VeGF in endothelial cells. We hypothesized that altered VeGFr2 expression in BiS-treated endothelial cells may account for these attenuated responses to VeGF. The affect of the BiS zoledronic acid (Zol) was investigated in human umbilical vein endothelial cells using confocal microscopy, Western blotting, real-time PCR and flow cytometry. VEGFR2 accumulated within the Zol-treated endothelial cells (p=0.0002), though not on the cell surface (p>0.05). Zol did not induce VEGFR2-specific mRNA (p>0.05). ZOL inhibited endothelial cell chemotaxis towards VeGF (p=0.001). VeGF stimulation significantly reduced the amount of VEGFR2 in the endothelial cells (p=0.01). This response to VeGF was reduced by Zol (p>0.05). The effects of Zol on endothelial cell migration, VeGFr2 protein expression and response to VeGF were attenuated by geranylgeranyl pyrophosphate. Twoand one-way anoVas with Tukey or dunnett's multiple comparison adjustments were used. The data suggest that Zol induces aberrant VeGFr2 accumulation. This is not likely due to the induction of mrna transcription, but rather to the disruption of the mevalonate pathway. Introduction nitrogen-containing bisphosphonates (BiS) are potent inhibitors of bone resorption and are used during the treatment of many diseases, including multiple myeloma and metastatic breast cancer (1,2). recent in vitro and in vivo studies have suggested that BiS also affect endothelial cell functions and angiogenesis. The BiS zoledronic acid (Zol) inhibits cytokine-stimulated endothelial cell proliferation and migration (3), suggesting that Zol inhibits cellular responses to cytokines. Furthermore, BiS affect angiogenesis in vivo. Zol inhibits testosterone-stimulated angiogenesis within the prostate in mice (4). in an experimental cervical cancer model, Zol increased endothelial cell apoptosis and subsequently reduced angiogenesis (5). BiS also altered vascular response to wound healing. Zol reduced the number of endothelial cells within alveolar bone after tooth extraction (6). Collectively, these findings suggest that ZOL is capable of inhibiting endothelial cell function and angiogenesis. The molecular mechanisms accounting for the aberrant responses to growth factors and cytokines caused by BiS remain largely unknown. VeGF interacts with many receptors, including VeGFr1/ fms-like tyrosine kinase (FlT)-1 and VeGFr2/Kdr/ fetal liver kinase (FlK)-1. experimental evidence supports VeGFr2 as the major receptor mediating cell signaling in endothelial cells in response to VeGF. Selective activation of VeGFr1 does not induce cell migration or intercellular calcium release in endothelial cells (7); furthermore, transgenic mice with VeGFr1 lacking the cytoplasmic portion had apparently normal vasculogenesis and angiogenesis (8). In vitro, VeGF stimulation induces minimal tyrosine VeGFr1 phosphorylation in endothelial cells (9). By contrast, the functional inactivation of VeGFr2 by a blocking antibody disrupted angiogenesis (10). endothelial cells expressed increased VeGFr2 mrna at the site of arterial injury (11). VeGF-stimulated endothelial cells phosphorylate VeGFr2 and downstream signaling targets, such as Plc-γ (12). These findings suggest that VEGFR2 is the main receptor responsible for signal transduction during VeGF-stimulation in endothelial cells. Therefore, we hypothesized that BiS modulate VeGFr2 expression in endothelial cells. Accumulation of VEGFR2 in zoledronic acid-treated endothelial cells daVid l. BaSi1, Soo Woon lee3, Sarah helFMan1, aMi MariaSh1 and ScoTT a. lunoS2 1division of oral and Maxillofacial Surgery, university of Minnesota dental School, Minneapolis, Mn 55455; 2Biostatistical design and analysis center, clinical and Translational Science institute, university of Minnesota, Minneapolis, Mn 55414, uSa; 3Busan Paik hospital, inje university, Busan 614-735, Korea received december 31, 2009; accepted March 8, 2010 doi: 10.3892/mmr_00000271 Correspondence to: dr david l. Basi, department of developmental and Surgical Science, division of oral and Maxillofacial Surgery, university of Minnesota dental School, 515 delaware Street Se, 7-174 Moos Tower, Minneapolis, Mn 55455,