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      Margatoxin Inhibits VEGF-Induced Hyperpolarization, Proliferation and Nitric Oxide Production of Human Endothelial Cells

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          Background: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K<sup>+</sup> channel, nitric oxide (NO) and Ca<sup>2+</sup> signaling was reported. We examined whether the K<sup>+</sup> channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC. Methods: Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca<sup>2+</sup> (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC (HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [<sup>3</sup>H]-thymidine incorporation (TI). Results: VEGF (5–50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml (n = 30, p < 0.05). This effect was completely blocked by MTX (5 µmol/l). VEGF caused an increase in transmembrane Ca<sup>2+</sup> influx (n = 30, p < 0.05) that was sensitive to MTX and the blocker of transmembrane Ca<sup>2+</sup> entry 2-aminoethoxydiphenyl borate (APB, 100 µmol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca<sup>2+</sup> (n = 30, p < 0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX (CC: –58%, TI –121%); APB (CC –99%, TI –187%); N-monomethyl- L-arginine (300 µmol/l: CC: –86%, TI –164%). Conclusions: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca<sup>2+</sup> entry that is responsible for the effects on endothelial proliferation and NO production.

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          Most cited references 17

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          Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid

           D Senger,  S Galli,  A. Dvorak (1983)
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            Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.

            Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis and angiogenesis. To investigate the role of nitric oxide (NO) in VEGF-induced proliferation and in vitro angiogenesis, human umbilical vein endothelial cells (HUVEC) were used. VEGF stimulated the growth of HUVEC in an NO-dependent manner. In addition, VEGF promoted the NO-dependent formation of network-like structures in HUVEC cultured in three dimensional (3D) collagen gels. Exposure of cells to VEGF led to a concentration-dependent increase in cGMP levels, an indicator of NO production, that was inhibited by nitro-L-arginine methyl ester. VEGF-stimulated NO production required activation of tyrosine kinases and increases in intracellular calcium, since tyrosine kinase inhibitors and calcium chelators attenuated VEGF-induced NO release. Moreover, two chemically distinct phosphoinositide 3 kinase (PI-3K) inhibitors attenuated NO release after VEGF stimulation. In addition, HUVEC incubated with VEGF for 24 h showed an increase in the amount of endothelial NO synthase (eNOS) protein and the release of NO. In summary, both short- and long-term exposure of human EC to VEGF stimulates the release of biologically active NO. While long-term exposure increases eNOS protein levels, short-term stimulation with VEGF promotes NO release through mechanisms involving tyrosine and PI-3K kinases, suggesting that NO mediates aspects of VEGF signaling required for EC proliferation and organization in vitro.
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              Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.

              Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                October 2005
                28 September 2005
                : 42
                : 5
                : 368-376
                Departments of aCardiology and Angiology, and bPhysiology, Justus Liebig University of Giessen, Giessen, Germany
                87159 J Vasc Res 2005;42:368–376
                © 2005 S. Karger AG, Basel

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                Page count
                Figures: 4, References: 38, Pages: 9
                Research Paper


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