Ali Erdogan a , Christian Alexander Schaefer a , Matthias Schaefer b , Doerte Wiebke Luedders a , Florian Stockhausen a , Yaser Abdallah b , Claudia Schaefer b , Astrid Kerstin Most a , Harald Tillmanns a , Hans Michael Piper b , Christoph Ruediger Wolfram Kuhlmann a
28 September 2005
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.