Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular growth and recruitment of perivascular mural cells. The purpose of the present study was to investigate the signalling events underlying the stimulation of vasculogenesis of mouse embryonic stem (ES) cells by PDGF-BB. PDGF-BB increased vascular sprouting and branching of capillary-like structures in embryoid bodies as evaluated by computer-assisted analysis of CD31-positive cell structures, and activated extracellular-regulated kinase 1,2 (ERK1,2) as well as c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase (MAPK) or PI3-kinase (PI3K). Microfluorometric analysis of fluo-4 fluorescence revealed that treatment with PDGF-BB raised intracellular calcium (Ca²⁺) in differentiating ES cells expressing the PDGF receptor-ß (PDGFR-ß), which was abolished in the presence of the intracellular Ca²⁺ chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) in embryoid bodies as evaluated using the redox-sensitive dye H2DCF-DA. ROS generation was blunted in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin as well as in the presence of BAPTA, suggesting that ROS generation is regulated by intracellular Ca2+ transients. The stimulation of vasculogenesis of ES cells upon treatment with PDGF-BB was significantly inhibited by the ERK 1,2 inhibitor U0126, the NADPH oxidase inhibitors DPI, apocynin, 4-(2-aminoethyl)benzene-sulfonylfluoride (AEBSF) and VAS2870, the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycin (NMPG) as well as by BAPTA.

Conclusion: 

Our data demonstrate that the pro-vasculogenic effects of PDGF-BB are mediated by Ca²⁺-induced ROS generation resulting in the activation of an ERK1,2-mediated signal transduction cascade.