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

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 [H-3]cGMP-radioimmunoassay. Phosphorylation of the p42/p44 MAP-kinase (MAPK) and the endothelial nitric oxide synthase (eNOS) was analyzed by immumocytochemistry. 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 mu mol L-1), or the MEK inhibitor PD 98059 (20 mu mol 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.