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Nitric oxide (NO) is a key regulator of cardiomyogenesis of embryonic stem (ES) cells. However, signaling pathways involving the energy sensor AMP-activated protein kinase (AMPK) and/or mammalian target of rapamycin (mTOR) resulting in NO generation and stimulation of cardiomyogenesis are currently not known. Herein, the role of AMPK- versus mTOR-regulated signaling pathways and the impact of NO for cardiomyogenesis of mouse ES cells were investigated. Activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAr) or metformin as well as inactivation of AMPK by compound C (Comp C), siRNA ablation of AMPKα2, or exogenous ATP stimulated cardiomyogenesis of ES cells. Inhibition of AMPK by Comp C resulted in phosphorylation of mTOR and generation of NO. NO generation was likewise achieved when AMPK was either activated by AICAr or mTOR was inhibited by rapamycin, suggesting that NO generation occurred by two mutually active parallel signaling pathways, one being AMPK dependent and mTOR independent (AICAr pathway) and the other being AMPK independent and mTOR dependent (Comp C pathway). Consequently, cardiomyogenesis as well as NO generation was completely abrogated when ES cells were cultivated in the presence of rapamycin and Comp C, which inhibit both signaling pathways. The impact of NO for cardiomyogenesis of ES cells was corroborated in experiments showing that the effects of Comp C on cardiomyogenesis of ES cells were abolished by the NO synthase inhibitors NG-monomethyl-l-arginine and N (G)-nitro-l-arginine methyl ester. In summary, our data demonstrate that NO generation downstream of AMPK and mTOR is activated by distinct, interacting signaling pathways that initiate cardiomyogenesis of ES cells. [ABSTRACT FROM AUTHOR]

AbstractThalidomide [α-(N-phthalimido)-glutarimide] exerts antiangiogenic properties and causes cardiac malformations in embryos. Herein the effects of thalidomide on cardiovascular differentiation were investigated in mouse embryonic stem (ES) cell-derived embryoid bodies. Thalidomide inhibited the formation of capillary-like blood vessels and decreased tumor-induced angiogenesis in confrontation cultures of embryoid bodies and multicellular prostate tumor spheroids, but stimulated cardiomyogenesis of ES cells. The number of CD31- and CD144-positive endothelial cells was not impaired, suggesting that thalidomide acted on vascular tube formation and cell migration rather than endothelial differentiation. Thalidomide increased reactive oxygen species generation, which was abolished by the NADPH oxidase inhibitor VAS2870 and the complex I respiratory chain inhibitor rotenone. Conversely, thalidomide decreased nitric oxide (NO) generation and endothelial NO synthase activity. VAS2870 abrogated thalidomide stimulation of cardiomyogenesis, whereas inhibition of vasculogenesis persisted. In NOX-1 and NOX-4 shRNA gene-inactivated ES cells, cardiomyogenesis was severely impaired and thalidomide failed to stimulate cardiac cell commitment. The NO donor S-nitrosopenicillamine reversed the antiangiogenic effect of thalidomide and increased capillary structure formation, whereas scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and inhibition of endothelial NO synthase by NG-nitro-l-arginine methyl ester decreased cardiovascular differentiation. Our data demonstrate that thalidomide causes an imbalance of reactive oxygen species/NO generation, thus stimulating cardiomyogenesis and impairing vascular sprout formation. Antioxid. Redox Signal.13, 1813–1827. [ABSTRACT FROM AUTHOR]

Electromagnetic fields (EMFs) are used to treat bone diseases. Herein, the effects of static EMFs on chondroosteogenesis and vasculogenesis of embryonic stem (ES) cells and bone mineralization of mouse fetuses were investigated. Treatment of differentiating ES cells with static EMFs (0.4–2 mT) stimulated vasculogenesis and chondro-osteogenesis and increased reactive oxygen species (ROS), which was abolished by the free radical scavengers trolox, 1,10-phenanthroline (phen), and the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI). In contrast, EMFs of 10 mT field strength exerted inhibitory effects on vasculogenesis and chondro-osteogenesis despite robust ROS generation. EMFs of 1 mT and 10 mT increased and decreased vascular endothelial growth factor (VEGF) expression, respectively, which was abolished by DPI and radical scavengers. EMFs activated extracellular-regulated kinase 1/2 (ERK1/2), p38, and c-jun N-terminal kinase (JNK), which was sensitive to DPI treatment. The increase in VEGF by EMFs was inhibited by the ERK1/2 inhibitor U0126 but not by SB203580 and SP600125, which are p38 and JNK inhibitors, respectively, suggesting VEGF regulation by ERK1/2. Chondroosteogenesis and vasculogenesis of ES cells was blunted by trolox, DPI, and the VEGF receptor-2 (flk-1) antagonist SU5614. In mouse fetuses 1 mT EMFs increased and 10 mT EMFs decreased bone mineralization, which was abolished in the presence of trolox. Hence, EMFs induced chondro-osteogenesis and vasculogenesis in ES cells and bone mineralization of mouse fetuses by a ROS-dependent up-regulation of VEGF expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]