Platelet-derived growth factor BB stimulates vasculogenesis of embryonic stem cell-derived endothelial cells by calcium-mediated generation of reactive oxygen species.
OBJECTIVE
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 is to investigate the signalling events underlying the stimulation of vasculogenesis of mouse embryonic stem (ES) cells by PDGF-BB.


RESULTS
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. It also activated extracellular-regulated kinase 1,2 (ERK1,2) and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinase or PI 3-kinase. Microfluorometric analysis of fluo-4 fluorescence revealed that treatment with PDGF-BB raised intracellular Ca(2+) levels in differentiating ES cells expressing the PDGF receptor beta, an effect that was abolished in the presence of the intracellular Ca(2+) chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) levels in embryoid bodies as evaluated using the redox-sensitive dye H(2)DCF-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 Ca(2+) transients. The stimulation of vasculogenesis of ES cells upon treatment with PDGF-BB was significantly inhibited by the ERK1,2 inhibitor U0126, the NADPH oxidase inhibitors DPI, apocynin, 4-(2-aminoethyl)benzenesulfonylfluoride and VAS2870, the free radical scavengers vitamin E, and N-(2-mercaptopropionyl)glycin as well as by BAPTA.


CONCLUSIONS
Our data demonstrate that the pro-vasculogenic effects of PDGF-BB are mediated by Ca(2+)-induced ROS generation, resulting in the activation of an ERK1,2-mediated signal transduction cascade.
Lange
Sabine
S
Department of Physiology, Justus-Liebig-University Giessen, Aulweg 129, 35392 Giessen, Germany.
Heger
Jaqueline
J
Euler
Gerhild
G
Wartenberg
Maria
M
Piper
Hans Michael
HM
Sauer
Heinrich
H
D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D000818:Animals; D000077214:Becaplermin; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D043925:Angiogenesis Inducing Agents; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D017382:Reactive Oxygen Species; D000077214:Becaplermin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium