A new signaling mechanism of hepatocyte growth factor‐induced endothelial proliferation

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 [3H]-cGMP-radioimmunoassay. Phosphorylation of the p42/p44 MAP-kinase (MAPK) and the endothelial nitric oxide synthase (eNOS) was analyzed by immunocytochemistry. Results: A dose-dependent (1–30 ng mL−1) increase of HUCVEC proliferation with a maximum at a concentration of 15 ng mL−1 was induced by HGF. This effect was significantly reduced by the addition of the K+ channel blocker iberiotoxin (100 nmol L−1), eNOS inhibitor l-NMMA (300 μmol L−1), or the MEK inhibitor PD 98059 (20 μmol L−1). 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 = 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.