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Summary. Background: The hepatocyte growth factor (HGF) has been shown to promote endothelial cell proliferation. In this study, the signaling cascade responsible for the HGF-induced proliferation was examined. Methods: The proliferation of human umbilical cord vein endothelial cells (HUCVEC) was determined using cell counts. Changes of the membrane potential were analyzed using the fluorescence dye DiBAC. Intracellular cGMP-levels were measured by means of [ ). A HGF-induced hyperpolarization that was blocked by iberiotoxin was observed. In addition, HGF-induced activation of the eNOS was blocked by the K + channel inhibitor. An increase of +101% MAPK phosphorylation was induced by HGF, which was blocked, if the cells were treated with L-NMMA (n ¼ 20; P < 0.05), whereas HGF-induced phosphorylation of the eNOS was not affected by MEK inhibition. Conclusions: Hepatocyte growth factor modulates endothelial K + channels causing an activation of the eNOS; the increase of nitric oxide is necessary for the phosphorylation of the MAPK inducing the proliferation of HUCVEC.

Apigenin-induced nitric oxide production involves calcium-activated potassium channels and is responsible for antiangiogenic effects. Summary. Background: The dietary flavonoid apigenin (Api) has been demonstrated to exert multiple beneficial effects upon the vascular endothelium. The aim of this study was to examine whether Ca 2+ -activated K + channels (K Ca ) are involved in endothelial nitric oxide (NO) production and antiangiogenic effects. Methods: Endothelial NO generation was monitored using a cyclic guanosine monophosphate radioimmunoassay. K Ca activity and changes of the intracellular Ca 2+ concentration [Ca 2+ ] i were analyzed using the fluorescent dyes bisbarbituric acid oxonol, potassium-binding benzofuran isophthalate, and fluo-3. The endothelial angiogenic parameters measured were cell proliferation, [ 3 H]-thymidine incorporation, and cell migration (scratch assay). Akt phosphorylation was examined using immunohistochemistry. Results: Api caused a concentration-dependent increase in cyclic guanosine monophosphate levels, with a maximum effect at a concentration of 1 lM. Api-induced hyperpolarization was blocked by the small and large conductance K Ca inhibitors apamin and iberiotoxin, respectively. Furthermore, apamin and iberiotoxin blocked the late, long-lasting plateau phase of the Api-induced biphasic increase of [Ca 2+ ] i . Inhibition of Ca 2+ signaling and the K Ca blockade both blocked NO production. Prevention of all three (NO, Ca 2+ , and K Ca signaling) reversed the antiangiogenic effects of Api under both basal and basic fibroblast growth factor-induced culture conditions. Basic fibroblast growth factor-induced Akt phosphorylation was also reduced by Api. Conclusions: Based on our experimental results we propose the following signaling cascade for the effects of Api on endothelial cell signaling. Api activates small and large conductance K Ca , leading to a hyperpolarization that is followed by a Ca 2+ influx. The increase of [Ca 2+ ] i is responsible for an increased NO production that mediates the antiangiogenic effects of Api via Akt dephosphorylation.

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