Cardiovascular Research Advance Access first published online on September 20, 2008
This version [Corrected Proof] published online on October 22, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn258
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Platelet-derived growth factor BB stimulates vasculogenesis of embryonic stem cell-derived endothelial cells by calcium-mediated generation of reactive oxygen species
1 Department of Physiology, Justus-Liebig-University Giessen, Aulweg 129, 35392 Giessen, Germany
2 Department of Internal Medicine I, Friedrich-Schiller-University Jena, Germany
* Corresponding author. Tel: +49 641 9947333; fax: +49 641 9947219. E-mail address: heinrich.sauer{at}physiologie.med.uni-giessen.de
Aims: 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.
Methods and 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 Ca2+ levels in differentiating ES cells expressing the PDGF receptor β, an effect that was abolished in the presence of the intracellular Ca2+ chelator BAPTA. Furthermore, PDGF-BB raised reactive oxygen species (ROS) levels 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 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.
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.
KEYWORDS Embryonic stem cell; Angiogenesis; Platelet-derived growth factor; Reactive oxygen species; Vasculogenesis
Time for primary review: 35 days
This paper was guest edited by Mark Post, Maastricht University.
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