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Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD) synthesis and is involved in cancer cell proliferation through regulation of energy production pathways. Therefore, NAMPT inhibitors are promising drugs for cancer therapy by limiting energy supply of tumours. Herein, we demonstrated that the NAMPT inhibitor FK866 ((E)-N-(4-(1-Benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) dose-dependently inhibited growth and cell motility of DU-145 prostate tumour spheroids and decreased the intracellular ATP concentration. The apoptosis marker cleaved caspase-3 remained unchanged, but the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) was upregulated. Growth inhibition was reversed upon co-administration of NAD to the cell culture medium. FK866 decreased calcein as well as pheophorbide A efflux from tumour spheroids and increased doxorubicin toxicity, indicating interference with function of drug efflux transporters. DU-145 multicellular tumour spheroids expressed the stem cell associated markers CD133, CD44, Oct4, Nanog, Sox2, and drug transporters ABCB1, ABCG2, and ABCC1 which are associated with stem cell properties in cancer cells. The ABCB1 inhibitor zosuquidar, the ABCG2 inhibitor Ko143, and the ABCC1 inhibitor MK571 increased calcein retention. Neither protein expression of stem cell markers, nor drug transporters was significantly changed upon FK866 treatment. In conclusion, our data suggest that FK866 inhibits prostate cancer cell proliferation by interference with the energy metabolism, and function of drug efflux transporters.

BackgroundOversupply of fatty acids (FAs) to cardiomyocytes (CMs) is associated with increased ceramide content and elevated the risk of lipotoxic cardiomyopathy. Here we investigate the role of ceramide accumulation on mitochondrial function and mitophagy in cardiac lipotoxicity using CMs derived from human-induced pluripotent stem cells (h-iPSCs). Methods and resultsMature CMs derived from h-iPSCs exposed to the diabetic-like environment or transfected with plasmids overexpressing serine-palmitoyltransferase long chain base subunit 1 (SPTLC1), a subunit of the serine-palmitoyltransferase (SPT) complex, resulted in increased intracellular ceramide levels. Accumulation of ceramides impaired insulin-dependent phosphorylation of Akt through activating protein phosphatase 2A (PP2A) and disturbed gene and protein levels of key metabolic enzymes including GLUT4, AMPK, PGC-1α, PPARα, CD36, PDK4, and PPARγ compared to controls. Analysis of CMs oxidative metabolism using a Seahorse analyzer showed a significant reduction in ATP synthesis-related O2 consumption, mitochondrial basal respiration and respiratory capacity, indicating an impaired mitochondrial function under diabetic-like conditions or SPTLC1-overexpression. Further, ceramide accumulation increased mitochondrial fission regulators such as dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF) as well as auto/mitophagic proteins LC3B and PINK-1 compared to control. Incubation of CMs with the specific SPT inhibitor (myriocin) showed a significant increase in mitochondrial fusion regulators the mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) as well as p-Akt, PGC-1 α, GLUT-4, and ATP production. In addition, a significant decrease in auto/mitophagy and apoptosis was found in CMs treated with myriocin.ConclusionsOur results suggest that ceramide accumulation has important implications in driving insulin resistance, oxidative stress, increased auto/mitophagy, and mitochondrial dysfunction in the setting of lipotoxic cardiomyopathy. Therefore, modulation of the de novo ceramide synthesis pathway may serve as a novel therapeutic target to treat metabolic cardiomyopathy.

Silibinin, the bioactive compound of milk thistle (Silybum marianum), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.

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Polyunsaturated fatty acids (PUFAs) and their metabolites may influence cell fate regulation. Herein, we investigated the effects of linoleic acid (LA) as ω-6 PUFA, eicosapentaenoic acid (EPA) as ω-3 PUFA and palmitic acid (PA) on vasculogenesis of embryonic stem (ES) cells. LA and EPA increased vascular structure formation and protein expression of the endothelial-specific markers fetal liver kinase-1, CD31 as well as VE-cadherin, whereas PA was without effect. LA and EPA increased reactive oxygen species (ROS) and nitric oxide (NO), activated endothelial NO synthase (eNOS) and raised intracellular calcium. The calcium response was inhibited by the intracellular calcium chelator BAPTA, sulfo-N-succinimidyl oleate which is an antagonist of CD36, the scavenger receptor for fatty acid uptake as well as by a CD36 blocking antibody. Prevention of ROS generation by radical scavengers or the NADPH oxidase inhibitor VAS2870 and inhibition of eNOS by L-NAME blunted vasculogenesis. PUFAs stimulated AMP activated protein kinase-α (AMPK-α) as well as peroxisome proliferator-activated receptor-α (PPAR-α). AMPK activation was abolished by calcium chelation as well as inhibition of ROS and NO generation. Moreover, PUFA-induced vasculogenesis was blunted by the PPAR-α inhibitor GW6471. In conclusion, ω-3 and ω-6 PUFAs stimulate vascular differentiation of ES cells via mechanisms involving calcium, ROS and NO, which regulate function of the energy sensors AMPK and PPAR-α and determine the metabolic signature of vascular cell differentiation.

Objectives Differentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. Methods and results Treatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. Conclusions Mechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB.

Man-made magnetic fields (MFs) may exert adverse effects on mammalian embryonic development. Herein, we analysed the effect of 10mT 50Hz sinusoidal (AC) or static (DC) MFs versus 1mT MFs on embryonic development of mice. Exposure for 20days during gestation to 10mT MFs increased resorptions and dead fetuses, decreased crown-rump length and fresh weight, reduced blood vessel differentiation and caused histological changes, accompanied with diminished vascular endothelial growth factor (VEGF) protein expression in several organs. In embryonic stem (ES) cell-derived embryoid bodies exposure towards 10mT MFs increased reactive oxygen species (ROS), decreased vascular marker as well as VEGF expression and enhanced apoptosis. In conclusion, our combined data from in vivo and in vitro experiments identified VEGF as an important mediator during embryonic development that can be influenced by high strength MFs, which in consequence leads to severe abnormalities in fetus organs and blood vessel formation.

Ca2+-activated potassium (KCa) channels of small and intermediate conductance influence proliferation, apoptosis, and cell metabolism. We analysed whether prolonged activation of KCa channels by zoxazolamine (ZOX) induces differentiation of mouse embryonic stem (ES) cells towards cardiomyocytes. ZOX treatment of ES cells dose-dependent increased the number and diameter of cardiac foci, the frequency of contractions as well as mRNA expression of the cardiac transcription factor Nkx-2.5, the cardiac markers cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), ventricular myosin light chain-2 (MLC2v), and the pacemaker hyperpolarization-activated, cyclic nucleotide-gated 4 channel (HCN4). ZOX induced hyperpolarization of membrane potential due to activation of IKCa, raised intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) in a Ca2+-dependent manner. The Ca2+ response to ZOX was inhibited by chelation of Ca2+ with BAPTA-AM, release of Ca2+ from intracellular stores by thapsigargin and the phospholipase C (PLC) antagonist U73,122. Moreover, the ZOX-induced Ca2+ response was blunted by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) as well as the specific P2Y1 antagonist MRS 2,179, suggesting purinergic receptor-stimulated signal transduction. Consequently, ZOX initiated ATP release from differentiating ES cells, which was inhibited by the chloride channel inhibitor NPPB and the gap junction inhibitor carbenoxolone (CBX). The stimulation of cardiomyogenesis by ZOX was blunted by the nitric oxide synthase (NOS) inhibitor l-NAME, as well as CBX and NPPB. In summary, our data suggest that ZOX enhances cardiomyogenesis of ES cells by ATP release presumably through gap junctional hemichannels, purinergic receptor activation and intracellular Ca2+ response, thus promoting NO generation.

The milk thistle compound Silibinin (i.e., a 1:1 mixture of Silybin A and Silybin B) stimulates vasculogenesis of mouse embryonic stem (ES) cells. Because vasculogenesis and leukopoiesis are interrelated, the effect of Silibinin on leukopoiesis of ES cells was investigated. Treatment of differentiating ES cells with hydrosoluble Silibinin-C-2',3-dihydrogen succinate dose-dependent increased the number of CD18+ , CD45+ , and CD68+ cells, indicating leukocyte/macrophage differentiation. Silibinin treatment activated phosphoinositide 3-kinase (PI3K), AKT (protein kinase B), signal transducer and activator of transcription 3 (STAT3), stimulated hypoxia-induced factor-1α (HIF-1α), and vascular endothelial growth factor receptor 2 (VEGFR2) expression and raised intracellular nitric oxide (NO). Western blot experiments showed that upon coincubation with either the PI3K inhibitor LY294002, the STAT3 inhibitor Stattic, the AKT antagonist AKT inhibitor VIII, or the NO inhibitor L-NAME, the Silibinin-induced expression of CD18, CD45, and CD68 was abolished. Moreover, the stimulation of HIF-1α and VEGFR2 expression was blunted upon STAT3 and PI3K/AKT inhibition. Treatment of differentiating ES cells with L-NAME abolished the stimulation of VEGFR2 and VE-cadherin expression achieved with Silibinin, indicating that NO is involved in vasculogenesis and leukocyte differentiation pathways. In summary, the data of the present study demonstrate that Silibinin stimulates leukocyte differentiation of ES cells, which is associated to vasculogenesis and regulated by PI3K/AKT-, STAT3-, and NO-mediated signaling.

The milk thistle (Silybum marianum (L.) Gaertn.) compound silibinin may be an inhibitor of the angiotensin II type 1 (AT1) receptor which is expressed in differentiating embryonic stem (ES) cells and is involved in the regulation of cardiomyogenesis. In the present study, it was demonstrated that silibinin treatment decreased the number of spontaneously contracting cardiac foci and cardiac cell areas differentiated from ES cells as well as contraction frequency and frequency of calcium (Ca2+) spiking. In contrast, angiotensin II (Ang II) treatment stimulated cardiomyogenesis as well as contraction and Ca2+ spiking frequency, which were abolished in the presence of silibinin. Intracellular Ca2+ transients elicited by Ang II in rat smooth muscle cells were not impaired upon silibinin treatment, excluding the possibility that the compound acted on the AT1 receptor. Ang II treatment activated extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways in embryoid bodies which were abolished upon silibinin pretreatment. In summary, our data suggest that silibinin inhibits cardiomyogenesis of ES cells by interfering with Ang II signaling downstream of the AT1 receptor.

Objective Cell injury releases nucleic acids supporting inflammation and stem cell activation. Here, the impact of extracellular ribonucleic acid, especially transfer RNA (ex-tRNA), on vasculogenesis and leukopoiesis of mouse embryonic stem (ES) cells was investigated. Approach and results ex-tRNA, whole cell RNA and ribosomal RNA (ex-rRNA) but not DNA increased CD31-positive vascular structures in embryoid bodies. Ex-tRNA and ex-rRNA increased numbers of VEGFR2+, CD31+ and VE-cadherin+ vascular cells as well as CD18+, CD45+ and CD68+ cells, indicating leukocyte/macrophage differentiation. This was paralleled by mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor-165 (VEGF165) and neuropilin 1 (NRP1), phosphorylation of phosphatidyl inositol 3-kinase (PI3K) and VEGF receptor 2 (VEGFR2) as well as mRNA expression of α-smooth muscle actin (α-SMA). ex-tRNA was taken up by endosomes, increased expression of the pro-angiogenic semaphorin B4 receptor plexin B1 as well as the ephrin-type B receptor 4 (EphB4) and ephrinB2 ligand and enhanced cell migration, which was inhibited by the VEGFR2 antagonist SU5614 and the PI3K inhibitor LY294002. This likewise abolished the effects of ex-tRNA on vasculogenesis and leukopoiesis of ES cells. Ex-tRNA increased NOX1, NOX2, NOX4 and DUOX2 mRNA and boosted the generation of superoxide and hydrogen peroxide which was inhibited by radical scavengers, the NADPH oxidase inhibitors apocynin, VAS2870, ML171, and plumbagin as well as shRNA silencing of NOX1 and NOX4. Conclusions Our findings indicate that ex-tRNA treatment induces vasculogenesis and leukopoiesis of ES cells via superoxide/hydrogen peroxide generated by NADPH oxidase and activation of VEGFR2 and PI3K.

The β-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that β-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific β-adrenergic receptor (β-adrenoceptor) antagonist Propranolol, the β1-adrenoceptor-specific antagonist Atenolol and the β2-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three β-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, β-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor 165 (VEGF165), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of β-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with β-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that β-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.

The plasticity of human adipose tissue-derived stem cells (hASCs) is promising, but differentiation in vitro toward endothelial cells is poorly understood. Flow cytometry demonstrated that hASCs isolated from excised fat tissue were positive for CD29, CD44, CD70, CD90, CD105, and CD166 and negative for the endothelial marker CD31, and the hematopoietic cell markers CD34 and CD133. hASCs differentiated into adipocytes after cultivation in adipogenic medium. Exposure of hASCs for 10 days under hypoxia (3% oxygen) in combination with leptin increased the percentage of CD31(+) endothelial cells as well as CD31, VE-Cadherin, Flk-1, Tie2, von Willebrand factor, and endothelial cell nitric oxide synthase mRNA expression. This was enhanced on co-incubation of vascular endothelial growth factor (VEGF) and leptin, whereas VEGF alone was not sufficient. Moreover, hASCs cultured on a matrigel surface under hypoxia/VEGF/leptin, showed a stable branching network. Hypoxic conditions significantly decreased apoptosis as evaluated by cleaved caspase-3, and increased prolyl hydroxylase domain 3 mRNA expression. Hypoxia increased expression of VEGF as well as leptin transcripts, which were significantly inhibited on co-incubation with either VEGF or leptin or a combination of both. Furthermore, leptin treatment of hypoxic cells increased the expression of the long/signaling form of the leptin receptor (ObRL), which was augmented on co-incubation with VEGF. The observed endothelial differentiation was dependent on the Akt pathway, as co-administration with Akt inhibitor abolished the observed effects. In conclusion, our data demonstrate that hASCs can be efficiently differentiated to endothelial cells by mimicking the hypoxic and pro-angiogenic microenvironment of adipose tissue.

α2-macroglobulin (α2M) is an acute-phase protein released upon challenges like cardiac hypertrophy and infarction. α2M signals via the low density lipoprotein receptor-related protein (LRP-1) and may induce stem cell activation. In the present study, the effects of α2M on vasculogenesis/angiogenesis and underlying signaling cascades were investigated in mouse embryonic stem (ES) cells. LRP-1 was expressed in ES cells and upregulated during differentiation. α2M dose dependently increased CD31-positive vascular structures in ES cell-derived embryoid bodies, the early cardiovascular markers isl-1, Nkx-2.5, and flk-1 as well as numbers of VE-cadherin and flk-1-positive cells, but downregulated α-smooth muscle actin. Enhancement of vasculogenesis/angiogenesis by α2M was abolished by the LRP-1 antagonist receptor-associated protein (RAP) and LRP-1 blocking antibody. Notably, α2M stimulated vascular growth in the chicken chorioallantois membrane assay, but not in a human umbilical vein endothelial cell spheroid model. α2M increased fibroblast growth factor-2 (FGF-2) protein expression, which was abolished by RAP, induced nitric oxide (NO) generation as determined by 4,5-diaminofluorescein diacetate microfluorometry, and activated nitric oxide synthase-3 (NOS-3) as well as extracellular-regulated kinase 1,2 (ERK1/2) and phosphatidyl inositol 3-kinase (PI3K). NO generation, the increase in FGF-2 expression, and the stimulation of vasculogenesis/angiogenesis by α2M were blunted by the NO synthase inhibitor L-NAME, the ERK1/2 inhibitor PD98059, and the PI3K inhibitor LY294002. Furthermore, vasculogenesis/angiogenesis by α2M was inhibited in the presence of the FGF receptor 1 antagonist SU5402. In conclusion, α2M stimulates endothelial and early cardiac, but not smooth muscle differentiation of ES cells through generation of NO, activation of ERK1/2 as well as PI3K, and induction of FGF-2 expression.

Embryonic stem (ES) cells can differentiate into various kinds of cells, such as endothelial and hematopoietic cells. In addition, some evidence suggests that inflammatory mediators such as leukotrienes (LTs), which include the 5-lipoxygenase (LOX) family, can regulate endothelial cell differentiation. In the present study, the eicosanoid precursor arachidonic acid (AA) stimulated vasculogenesis of ES cells by increasing the number of fetal liver kinase-1+ vascular progenitor cells as well as vascular structures positive for platelet endothelial cell adhesion protein-1 and vascular endothelial cadherin. The stimulation of vasculogenesis and expression of the rate-limiting enzyme in the LT signaling pathway, 5-LOX-activating protein (FLAP), was blunted upon treatment with the FLAP inhibitors AM643 and REV5901. Vasculogenesis was significantly restored upon exogenous addition of LTs. Downstream of FLAP, the LTB4 receptor (BLT1) blocker U75302, the BLT2 receptor blocker LY255283 as well as the cysteinyl LT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased reactive oxygen species (ROS) generation, which was not affected upon either FLAP or cyclooxygenase-2 inhibition. Prevention of ROS generation by either the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycine or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and blunted the provasculogenic effect of AA. In summary, our data demonstrate that proinflammatory AA stimulates vasculogenesis of ES cells via the LT pathway by mechanisms involving ROS generation.

To date, the precise role of ion channels in developmental processes has remained elusive, although it is well accepted that cell differentiation and maturation affect the expression patterns of ion channels [1]. Calcium-activated potassium channels (SKCas) exhibit small (SK1-3, Kcnn1-3) or intermediate (SK4, IK, Kcnn4) unitary conductance for Stem Cell Rev and Rep (2012) 8:720–740 DOI 10.1007/s12015-011-9324-9

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.

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1(+)) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.

Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca2+]i which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca2+]i oscillations were dependent on extracellular Ca2+ and Ca2+ release from intracellular stores since they were abolished in Ca2+-free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP3 pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca2+]i oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca2+]i oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca2+]i oscillations in undifferentiated ASCs by controlling gap-junctional coupling. J. Cell. Physiol. 226: 1642–1650, 2011. © 2010 Wiley-Liss, Inc.

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.

Embryonic stem (ES) cells spontaneously differentiate capillary-like structures as well as leucocytes such as monocytes/macrophages, neutrophils, natural killer (NK) cells and cytototoxic T lymphocytes. The interplay between vasculogenesis and leucocyte differentiation as well as the population of tumour tissues with ES cell-derived leucocytes and endothelial cells is, however, not sufficiently specified. In the present study, gene expression of the cell surface markers CD68 and CD14 (expressed on monocytes and macrophages), Mac-1 (CD11b) (expressed on granulocytes, monocytes and NK cells) and CD16 (expressed on neutrophils) was investigated in murine CGR8 ES cells in relation to the endothelial cell markers CD31 and vascular endothelial (VE)-cadherin. Expression of leucocyte markers increased from day 7–8 of cell culture on. Furthermore, addition of macrophage colony-stimulating factor to the cell culture medium resulted in a threefold increase in the number of CD68+ monocytes/macrophages. Treatment of embryoid bodies with lipopolysaccharide (LPS) up-regulated CD14 thus suggesting functionality of the CD14 LPS receptor. Differentiation of vascular structures positive for CD31 and VE-cadherin preceded leucocyte differentiation by 2 days (i.e. from day 5–6 on) suggesting that vasculogenesis may be a determinant of leucocyte differentiation. Consequently the Flk-1 antagonist SU5416 which inhibits vasculogenesis of ES cells significantly blunted leucocyte differentiation. Confrontation culture of embryoid bodies with multicellular breast tumour spheroids initiated significant increase of leucocyte cell numbers and invasion of leucocytes into the tumour tissue. In summary our data demonstrate that during ES cell differentiation vasculogenesis precedes leucocyte differentiation, and point towards the direction that leucocyte cell invasion into tumour tissue may initiate the pro-inflammatory microenvironment necessary for tumour vascularization.

Ca2+ oscillations have been considered to obey deterministic dynamics for almost two decades. We show for four cell types that Ca2+ oscillations are instead a sequence of random spikes. The standard deviation of the interspike intervals (ISIs) of individual spike trains is similar to the average ISI; it increases approximately linearly with the average ISI; and consecutive ISIs are uncorrelated. Decreasing the effective diffusion coefficient of free Ca2+ using Ca2+ buffers increases the average ISI and the standard deviation in agreement with the idea that individual spikes are caused by random wave nucleation. Array-enhanced coherence resonance leads to regular Ca2+ oscillations with small standard deviation of ISIs.

Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments.

Peroxisome proliferator-activated receptors (PPARα, -β and -γ) are nuclear receptors involved in transcriptional regulation of lipid and energy metabolism. Since the energy demand increases when cardiac progenitor cells are developing rhythmic contractile activity, PPAR activation may play a critical role during cardiomyogenesis of embryonic stem (ES) cells. It is shown that ES cells express PPARα, -β, and -γ mRNA during differentiation of ES cells towards cardiac cells. Treatment with PPARα agonists (WY14643, GW7647, and ciprofibrate) significantly increased cardiomyogenesis and expression of the cardiac genes MLC2a, ANP, MHC-β, MLC2v, and cardiac α-actin. Furthermore, WY14643 increased PPARα gene expression and the expression of the cardiogenic transcription factors GATA-4, Nkx2.5, DTEF-1, and MEF 2C. In contrast, the PPARα antagonist MK886 decreased cardiomyogenesis, whereas the PPARβ agonist L-165,041 as well as the PPARγ agonist GW1929 were without effects. Treatment with PPARα, but not PPARβ, and PPARγ agonists and MK886, resulted in generation of reactive oxygen species (ROS), which was inhibited in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin and the free radical scavengers vitamin E and N-(2-mercapto-propionyl)-glycine (NMPG), whereas the mitochondrial complex I inhibitor rotenone was without effects. The effect of PPARα agonists on cardiomyogenesis of ES cells was abolished upon preincubation with free radical scavengers and NADPH oxidase inhibitors, indicating involvement of ROS in PPARα, mediated cardiac differentiation. In summary, our data indicate that stimulation of PPARα but not PPARβ and -γ enhances cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase activity. Disclosure of potential conflicts of interest is found at the end of this article.

The prognosis of cancer disease is worsened upon shedding of tumor cells from the primary tumor, which escape to the blood stream and form metastases at distant sites within the body. Inhibition of cell shedding from the primary tumor could therefore be exploited to avoid metastasis and delay the progression of the cancer disease. In the present study, we investigated the effects of the polyphenols resveratrol, baicalein, epicatechin, epigallocatechin and polyphenon 60 on cell shedding from multicellular tumor spheroids of the murine mammacarcinoma cell line 4T1, cell invasion into embryonic stem cell-derived tissues, generation of reactive oxygen species (ROS) and expression of matrix metalloproteinase 9 (MMP-9). With increasing tumor spheroid growth MMP-9 expression was upregulated and cells detached from tumor spheroids and formed subspheroids that displayed pronounced ROS generation. Upon incubation with polyphenols tumor growth was arrested and cell shedding was totally abolished. Polyphenol treatment decreased ROS generation and downregulated MMP-9 expression. Furthermore, polyphenols significantly inhibited invasion of tumor cells into embryonic stem cell-derived, vascularized tissues. Our data suggest, that polyphenols inhibit cell shedding and invasion by their anti-oxidative capacity and downregulation of MMP-9 expression.

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5'-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5'-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca(2+)](i)) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca(2+)](i) transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca(2+)](i) transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca(2+)](i) response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca(2+)](i) transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Background Cardiotrophin-1 (CT-1) controls cardiomyogenesis of mouse embryonic stem (ES) cells. Objectives To investigate the signaling pathway underlying the action of CT-1 on cardiac cell differentiation. Methods Protein expression was analyzed by western blot technique and cardiac areas by immunohistochemistry. Calcium, reactive oxygen species (ROS) and nitric oxide (NO) were assessed by microfluorometry using fluo-4, H 2 DCF, and DAF-2DA, respectively. Gene inactivation of CT-1 was achieved by siRNA technology. Results CT-1 as well as its receptor gp 130 were transiently upregulated during differentiation of ES cells. Exogenous CT-1 enhanced cardiomyogenesis, increased the cardiac transcription factors MEF2c, Nkx-2.5, TEAD3 and GATA4, the cardiac proteins α-actinin, MLC2a, MYH7, MLC1a, MLC2v and HCN4 as well as vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor-2 (FGF-2) and atrial natriuretic peptide (ANP). CT-1 downregulation by small interfering RNA (siRNA) inhibited cardiomyogenesis and decreased VEGF, PDGF-BB, FGF-2 and ANP expression. CT-1 raised intracellular calcium which was abolished by the intracellular calcium chelator BAPTA, AM and thapsigargin. Moreover, CT-1 treatment increased ROS, followed by NO generation and NOS3 activation. During ES cell differentiation CT-1 was translocated to the cell nucleus. Exogenous CT-1 induced nuclear translocation of endogenous CT-1, which was inhibited by BAPTA, the NOS inhibitor L-N G -Nitroarginine methyl ester (l-NAME), the radical scavenger N-(2-mercaptopropionyl)-glycine (NMPG) as well as the janus kinase 2 (JAK2) inhibitor AG490 and the PI3 kinase (PI3K) inhibitor LY294002. Conclusions Nuclear translocation of CT-1 regulates cardiomyogenesis of ES cells and involves calcium, NO, ROS as well as CT-1 regulated signaling pathways.

Objective Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC). Methods and results PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229±9% and 362±54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 μM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF promoted a 4.2±0.2-fold increase of VSMC migration (modified Boyden chamber, p

Cardiomyogenesis in differentiating mouse embryonic stem (ES) cells is promoted by cardiotrophin-1 (CT-1), a member of the IL-6 interleukin superfamily that acts through the tall gp130 cytokine receptor. We show that prooxidants (menadione, hydrogen peroxide) as well as chemical (CoCl2) and physiological (1% O2) hypoxia increased CT-1 as well as HIF-1α protein and mRNA expression in embryoid bodies, indicating that CT-1 expression is regulated by reactive oxygen species (ROS) and hypoxia. Treatment with either prooxidants or chemical hypoxia increased gp130 phosphorylation and protein expression of NADPH oxidase subunits p22-phox, p47-phox, p67-phox, as well as Nox1 and Nox4 mRNA. Consequently, inhibition of NADPH oxidase activity by diphenylen iodonium chloride (DPI) and apocynin abolished prooxidant- and chemical hypoxia-induced upregulation of CT-1. Prooxidants and chemical hypoxia activated ERK1,2, JNK and p38 as well as PI3-kinase. The proxidant- and CoCl2-mediated upregulation of CT-1 was significantly inhibited in the presence of the ERK1,2 antagonist UO126, the JNK antagonist SP600125, the p38 antagonist SKF86002, the PI3-kinase antagonist LY294002, the Jak-2 antagonist AG490 as well as in the presence of free radical scavengers. Moreover, developing embryoid bodies derived from HIF-1α-/- ES cells lack cardiomyogenesis, and prooxidants as well as chemical hypoxia failed to upregulate CT-1 expression. Our results demonstrate that CT-1 expression in ES cells is regulated by ROS and HIF-1α and imply a crucial role of CT-1 in the survival and proliferation of ES-cell-derived cardiac cells.

Active Ca2+ transport in living cells necessitates controlled supply of metabolic energy. Direct coupling between sarco/endoplasmic reticulum (ER) Ca2+ ATPases (SERCA) and intracellular energy-generation sites has been well established experimentally. On the basis of these experimental findings we propose a pump-driven model to investigate complex dynamic properties of a cell system. The model describes the pump process both by the Ca2+ ATPase itself and by a suitable description of the glycolysis. The associated set of differential equations shows a rich behavior, the solutions ranging from simple periodic oscillations to complex patterns such as bursting and spiking. Recent experimental results on calcium oscillations in Xenopus laevis oocytes and on dynamic patterns of intracellular Ca2+ concentrations in electrically non-excitable cells are well described by corresponding theoretical results derived within the proposed model. The simulation results are further compared to spontaneous [Ca2+] oscillations in primitive endodermal cells.

Expression of the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) has been demonstrated to be regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) and inhibited by intracellular reactive oxygen species (ROS). Herein, P-gp and HIF-1alpha expression were investigated in multicellular prostate tumor spheroids overexpressing the ROS-generating enzyme Nox-1 in comparison to the mother cell line DU-145. In Nox-1-overexpressing tumor spheroids (DU-145Nox1) generation of ROS as well as expression of Nox-1 was significantly increased as compared to DU-145 tumor spheroids. ROS generation was significantly inhibited in the presence of the NADPH-oxidase antagonists diphenylen-iodonium chloride (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Albeit growth kinetic of DU-145Nox1 tumor spheroids was decreased as compared to DU-145 spheroids, elevated expression of Ki-67 was observed indicating increased cell cycle activity. In DU-145Nox1 tumor spheroids, expression of HIF-1alpha as well as P-gp was significantly decreased as compared to DU-145 spheroids, which resulted in an increased retention of the anticancer agent doxorubicin. Pretreatment with the free radical scavengers vitamin E and vitamin C increased the expression of P-gp as well as HIF-1alpha in Nox-1-overexpressing cells, whereas no effect of free radical scavengers was observed on mdr-1 mRNA expression. In summary, the data of the present study demonstrate that the development of P-gp-mediated MDR is abolished under conditions of elevated ROS levels, suggesting that the MDR phenotype can be circumvented by modest increase of intracellular ROS generation.

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of pre-B cells and prevents further development. We studied whether inhibition of BCR-ABL1 kinase activity using STI571 can relieve this differentiation block. STI571 treatment of leukemia patients induced expression of the Ig L chain-associated transcription factors IRF4 and SPIB, up-regulation of RAG1 and RAG2, Cκ and Cλ germline transcription, and rearrangement of Ig κ L chain (IGK) and Ig λ L chain (IGL) genes. However, STI571-treated pre-B ALL cells expressed λ L, but almost no κ L chains. This could be explained by STI571-induced rearrangement of the κ-deleting element (KDE), which can delete productively rearranged Vκ-Jκ joints. Amplifying double-strand breaks at recombination signal sequences within the IGK, KDE, and IGL loci revealed a coordinated sequence of rearrangement events induced by STI571: recombination of IGK gene segments was already initiated within 1 h after STI571 treatment, followed by KDE-mediated deletion of Vκ-Jκ joints 6 h later and, ultimately, IGL gene rearrangement after 12 h. Consistently, up-regulation of Cκ and Cλ germline transcripts, indicating opening of IGK and IGL loci, was detected after 1 and 6 h for IGK and IGL, respectively. Continued activity of the recombination machinery induced secondary IGK gene rearrangements, which shifted preferential usage of upstream located Jκ- to downstream Jκ-gene segments. Thus, inhibition of BCR-ABL1 in pre-B ALL cells 1) recapitulates early B cell development, 2) directly shows that IGK, KDE, and IGL genes are rearranged in sequential order, and 3) provides a model for Ig L chain gene regulation in the human.

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of human pre–B cells. Comparing genome-wide gene expression profiles of BCR-ABL1+ pre–B ALL and normal bone marrow pre–B cells by serial analysis of gene expression, many genes involved in pre–B cell receptor signaling are silenced in the leukemia cells. Although normal pre–B cells are selected for the expression of a functional pre–B cell receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region gene are unresponsive to pre–B cell receptor engagement and exhibit autonomous oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and differentiate into immature B cells expressing immunoglobulin light chains. BCR-ABL1 kinase activity is linked to defective pre–B cell receptor signaling and the expression of a truncated isoform of the pre–B cell receptor–associated linker molecule SLP65. Also in primary leukemia cells, truncated SLP65 is expressed before but not after treatment of the patients with STI571. We conclude that inhibition of BCR-ABL1 reconstitutes selection for leukemia cells expressing a functional (pre–) B cell receptor.

Function and regulation of the intrinsic prion protein (PrPc) are largely unknown. In the present study the regulation of PrPc expression by growth factors and cytokines that increase intracellular reactive oxygen species (ROS) levels was studied in glioma and neuroblastoma cells grown as multicellular tumor spheroids. PrPc protein was significantly increased when glioma spheroids were treated with either ATP, nerve growth factor (NGF), epidermal growth factor (EGF), or tumor necrosis factor alpha (TNF-alpha), whereas mRNA levels as evaluated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) remained unchanged. ATP, NGF, EGF, and TNF-alpha raised intracellular ROS levels as evaluated using the redox-sensitive fluorescence dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). The observed elevation in PrPc was completely abolished in the presence of the free radical scavengers vitamin E and ebselen, as well as following pretreatment with the NADPH-oxidase inhibitor diphenylen iodonium chloride (DPI), indicating that PrPc levels are regulated by intracellular ROS. The correlation of PrPc expression to the intracellular ROS levels was investigated by the use of neuroblastoma cells overexpressing either mutant V210I PrP, or wild-type PrPc. It was observed that the intracellular redox state was significantly reduced in PrPc as well as V210I PrP overexpressing cells as compared to non-transfected cells. Consequently, the observed elevation of ROS following treatment with ATP was completely abolished in PrP overexpressing cells. Our data are in line with the assumption that PrPc plays a role as free radical scavenger and/or sensor molecule for oxidative stress.

BACKGROUND The anticonvulsant valproic acid (VPA) exerts teratogenic properties and has been demonstrated to cause neural tube defects and malformations of the heart. The effect of VPA on the differentiation of cardiomyocytes from pluripotent murine embryonic stem cells (ES cells) was investigated. METHODS Embryoid bodies derived from ES cells were treated with different concentrations of VPA and the differentiation of cardiomyocytes was monitored by immunohistochemical staining for sarcomeric α-actinin. Cytotoxicity was evaluated by the use of the dead cell stain SYTOX green. Intracellular levels of reactive oxygen species (ROS) within the tissue were evaluated by the use of the redox-sensitive dye dichlorodihydrofluorescein diacetate (H2DCFDA). RESULTS VPA retarded the growth of ES cell-derived embryoid bodies but did not exert cytotoxic effects. The compound dose-dependently inhibited the development of spontaneously beating clusters of cardiomyocytes within embryoid bodies grown from ES cells and reduced the extension of beating areas of cardiac cells. Furthermore, VPA significantly increased ROS levels, indicating that VPA altered the intracellular redox balance. To investigate whether the inhibition of cardiomyocyte differentiation by VPA was owing to increased ROS overwhelming the intracellular antioxidative defense, the compound was coadministered with the free radical scavenger vitamin E. CONCLUSIONS This treatment significantly restored cardiomyogenic differentiation, indicating that VPA inhibits cardiomyogenesis of ES cells by increasing intracellular ROS levels. Birth Defects Research (Part A) 67:174–180, 2003. © 2003 Wiley-Liss, Inc.

Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance transporter P-glycoprotein (P-gp) has not been investigated. Herein, we demonstrate that with increasing size of DU-145 prostate multicellular tumor spheroids the pericellular oxygen pressure and the generation of reactive oxygen species decreased, whereas the alpha-subunit of HIF-1 (HIF-1alpha) and P-gp were up-regulated. Furthermore, P-gp was up-regulated under experimental physiological hypoxia and chemical hypoxia induced by either cobalt chloride or desferrioxamine. The pro-oxidants H2O2 and buthionine sulfoximine down-regulated HIF-1alpha and P-gp, whereas up-regulation was achieved with the radical scavengers dehydroascorbate, N-acetylcysteine, and vitamin E. The correlation of HIF-1alpha and P-gp expression was validated by the use of hepatoma tumor spheroids that were either wild type (Hepa1) or mutant (Hepa1C4) for aryl hydrocarbon receptor nuclear translocator (ARNT), i.e., HIF-1beta. Chemical hypoxia robustly increased HIF-1alpha as well as P-gp expression in Hepa1 tumor spheroids, whereas no changes were observed in Hepa1C4 spheroids. Hence, our data demonstrate that expression of P-gp in multicellular tumor spheroids is under the control of HIF-1.

Tumor-induced angiogenesis is a prerequisite for excessive tumor growth. Blood vessels invade the tumor tissue after degradation of the extracellular matrix scaffold by matrix metalloproteinases (MMPs). Inhibition of MMPs has been therefore suggested to be a useful tool to abolish neoangiogenesis of solid tumors. In the present study, antioxidative plant ingredients used in traditional Chinese medicine were investigated for their capacity to down-regulate MMP expression and to inhibit angiogenesis in embryonic stem cell-derived embryoid bodies and tumor-induced angiogenesis in confrontation cultures consisting of embryoid bodies and multicellular DU-145 prostate tumor spheroids. Embryoid bodies transiently expressed MMP-1, MMP-2, and MMP-9 during the time of differentiation of capillary-like structures. In confrontation cultures, MMP expression was increased compared with control tumor spheroids and embryoid bodies cultivated separately. The increased expression of MMPs in confrontation cultures was a result of elevated levels of reactive oxygen species (ROS) upon confrontation culture and was totally abolished in the presence of the free radical scavenger vitamin E. Incubation of embryoid bodies with baicalein, epicatechin, berberine, and acteoside, which are herbal ingredients used in traditional Chinese medicine, significantly inhibited angiogenesis in embryoid bodies and decreased intracellular ROS levels. Tumor-induced angiogenesis in confrontation cultures was totally abolished in the presence of the free radical scavenger vitamin E. Because herbal ingredients down-regulated MMP expression, we conclude that ROS generated during confrontation culture induce the expression of MMPs that are necessary for endothelial cell invasion into the tumor tissue.

It has been demonstrated that adenosine 5'-triphosphate (ATP) is actively secreted by cells, thereby eliciting Ca(2+)-dependent signal transduction cascades in an autocrine and paracrine manner. In the present study the effects of direct current (DC) electrical fields on ATP release, the intracellular Ca(2+) concentration [Ca(2+)](i) and growth of multicellular prostate tumor spheroids were investigated. Treatment of multicellular tumor spheroids by a single DC electrical field pulse with a field strength of 750 Vm(-1) for 60 seconds resulted in a transient Ca(2+) response, activation of c-Fos and growth stimulation. The initial [Ca(2+)](i) signal was elicited at the anode-facing side of the spheroid and spread with a velocity of approximately 12 microm per second across the spheroid surface. The electrical-field-evoked Ca(2+) response as well as c-Fos activation and growth stimulation of tumor spheroids were inhibited by pretreatment with the anion channel blockers NPPB, niflumic acid and tamoxifen. Furthermore, the Ca(2+) response elicited by electrical field treatment was abolished following purinergic receptor desensitivation by repetitive treatment of tumor spheroids with ATP and pretreatment with the purinergic receptor antagonist suramin as well as with apyrase. Electrical field treatment of tumor spheroids resulted in release of ATP into the supernatant as evaluated by luciferin/luciferase bioluminescence. ATP release was efficiently inhibited in the presence of anion channel blockers. Our data suggest that electrical field treatment of multicellular tumor spheroids results in ATP release, which concomitantly activates purinergic receptors, elicits a Ca(2+) wave spreading through the tumor spheroid tissue and stimulates tumor growth.

The effects of cell cycle inhibition on the expression of the multidrug resistance transporter P-glycoprotein (P-gp) as well as of the cyclin-dependent kinase (CDK) inhibitors p27 Kip1 and p21 WAFˇ1 were investigated in DU-145 prostate tumor spheroids. With increasing spheroid size the number of cells in the G0/G1 phase augmented, whereas the number of cells in the G2/M phase and the S phase of the cell cycle declined. The number of G0/G1 cells was elevated after incubation with either mimosine, staurosporine or serum-free medium. Mitomycin C and roscovitine increased the number of S phase cells. Roscovitine additionally increased cells in the G2/M phase. Incubation in serum-free medium upregulated p21 WAFˇ1 , p27 Kip1 and P-gp. Mimosine treatment resulted in upregulation of p27 Kip1 and P-gp, whereas p21 WAFˇ1 remained unchanged. Upon roscovitine treatment p27 Kip1 and p21 WAFˇ1 were downregulated, whereas P-gp was unaltered. Mitomycin C treatment resulted in downregulation of p27 Kip1 and p21 WAFˇ1 ; no significant change in P-gp levels was observed. Staurosporine induced upregulation of p21 WAFˇ1 whereas p27 Kip1 remained unaltered. P-gp was downregulated upon staurosporine treatment, which was owing to an elevation of intracellular reactive oxygen species by this compound. It is concluded that upregulation of P-gp in G0/G1 phase cells requires coexpression of the CDK inhibitor p27 Kip1 but not the CDK inhibitor p21 WAFˇ1 . fl 2002 Elsevier Science B.V. All rights reserved.

The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1(+) (Flk-1(+)) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1(+) cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration.

Intrinsic expression of the multidrug resistance (MDR) transporter P-glycoprotein (Pgp) may be regulated by reactive oxygen species (ROS). A transient expression of Pgp was observed during the growth of multicellular tumor spheroids. Maximum Pgp expression occurred in tumor spheroids with a high percentage of quiescent, Ki-67-negative cells, elevated glutathione levels, increased expression of the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF-1 as well as reduced ROS levels and minor activity of the mitogen-activated kinase (MAPK) members c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase ERK1,2, and p38 MAPK. Raising intracellular ROS by depletion of glutathione with buthionine sulfoximine (BSO) or glutamine starvation resulted in down-regulation of Pgp and p27Kip1, whereas ERK1,2 and JNK were activated. Down-regulation of Pgp was furthermore observed with low concentrations of hydrogen peroxide and epidermal growth factor, indicating that ROS may regulate Pgp expression. The down-regulation of Pgp following BSO treatment was abolished by agents interfering with receptor tyrosine kinase signaling pathways, i.e. the protein kinase C inhibitors bisindolylmaleimide I (BIM-1) and Ro-31-8220, the p21ras farnesyl protein transferase inhibitor III, the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1,2 activation. ROS involved as second messengers in receptor tyrosine kinase signaling pathways may act as negative regulators of Pgp expression.

Reactive oxygen species (ROS) are generated following ligand-receptor interactions and function as specific second messengers in signaling cascades involved in cell proliferation and differentiation. Although ROS are generated intracellularly by several sources, including mitochondria, the primary sources of ROS involved in receptor-mediated signaling cascades are plasma membrane oxidases, preferentially NADPH oxidases, with a rapid kinetics of activation and inactivation. This allows a tight up- and downregulation of intracellular ROS levels within the short time required for the transduction of signals from the plasma membrane to the cell nucleus. The mode of action of ROS may involve direct interaction with specific receptors, and/or redox-activation of members of signaling pathways such as protein kinases, protein phosphatases, and transcription factors. Furthermore, ROS act in concert with intracellular Ca(2+) in signaling pathways which regulate the balance of cell proliferation versus cell cycle arrest and cell death. The delicate intracellular interplay between oxidizing and reducing equivalents allows ROS to function as second messengers in the control of cell proliferation and differentiation.

Thalidomide is a teratogen with anti-angiogenic properties and causes stunted limb growth (dysmelia) during human embryogenesis. The molecular mechanisms of thalidomide action in embryopathy are currently unknown. Using the endothelial-specific antigen platelet endothelial cell adhesion molecule-1 and confocal laser scanning microscopy we have demonstrated that thalidomide exerts anti-angiogenic effects on the development of capillary structures in embryoid bodies differentiated from murine embryonic stem cells. Consequently, in thalidomide-treated embryoid bodies the diffusion properties of the tissue were deteriorated. Thalidomide raised reactive oxygen species (ROS), as revealed using 2'7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) as an indicator. A comparable ROS generation was achieved with the thalidomide hydrolysis product phthaloyl glutamic acid (PGA), but not with phthalimide (PI), the major component of thalidomide. ROS formation by thalidomide was inhibited by the hydroxyl radical scavengers mannitol and 2-mercaptoethanol. After coadministration of either 2-mercaptoethanol or mannitol with thalidomide the anti-angiogenic effects of thalidomide were abolished and the diffusion properties of the tissue were restored to the control values. In summary, our data suggest that thalidomide exerts its anti-angiogenic properties via the generation of toxic hydroxyl radicals, which impair vasculogenesis and angiogenesis during embryoid body development.

The effects of electromagnetic fields (EMFs) on the differentiation of cardiomyocytes in embryoid bodies derived from pluripotent embryonic stem (ES) cells were investigated. A single direct current (DC) field pulse was applied to 4-day-old embryoid bodies. The electrical field induced a hyperpolarization of the anode-facing side of embryoid bodies and a depolarization at the cathode-facing side. Significant effects of a single electrical field pulse applied for 90 s on cardiomyocyte differentiation were achieved with field strengths of 250 and 500 V/m, which increased both the number of embryoid bodies differentiating beating foci of cardiomyocytes and the size of the beating foci. The 500-V/m electrical field increased intracellular reactive oxygen species (ROS), but not [Ca(2+)](i) and activated nuclear factor kappa B (NF-kappaB). A comparable increase in the number of beating embryoid bodies was achieved by an incubation for 1 h with H(2)O(2) (1-10 nM), indicating that the electrical field effect was transduced via the intracellular generation of ROS. Because the radical scavengers dehydroascorbate and pyrrolidinedithiocarbamate (APDC) and the NF-kappaB antagonist N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibited cardiac differentiation, we assume that ROS and NF-kappaB may play a role in early cardiac development.

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.