Oxidative stress and regulation of adipogenic differentiation capacity by sirtuins in adipose stem cells derived from female patients of advancing age.

Patient age is critical for mesenchymal stem cell quality and differentiation capacity. We demonstrate that proliferation and adipogenic capacity of subcutaneous adipose stem cells (ASCs) from female patients declined with advanced age, associated with reduction in cell nucleus size, increase in nuclear lamina protein lamin B1/B2, and lamin A, upregulation of senescence marker p16INK4a and senescence-associated ß-galactosidase activity. Adipogenic induction resulted in differentiation of adipocytes and upregulation of adipogenic genes CCAAT enhancer binding protein alpha, fatty acid binding protein 4, lipoprotein lipase, and peroxisome proliferator-activated receptor-γ, which was not affected by the Sirt-1 activator YK-3-237 or the Sirt-1 inhibitor EX-527. Protein expression of the stem cell markers Oct4 and Sox2 was not significantly downregulated with advanced patient age. Mitochondrial reactive oxygen species were increased in ASCs from old-aged patients, whereas protein expression of NADPH oxidases NOX1 and NOX4 was downregulated, and dual oxidase isoforms remained unchanged. Generation of nitric oxide and iNOS expression was downregulated. Protein expression of Sirt-1 and Sirt-3 decreased with patient age, whereas Sirt-2 and Sirt-5 remained unchanged. Induction of adipogenesis stimulated protein expression of Sirt-1 and Sirt-3, which was not affected upon pre-incubation with the Sirt-1-activator YK-3-237 or the Sirt-1-inhibitor EX-527. The Sirt-1 inhibitor Sirtinol downregulated adiponectin protein expression and the number of adipocytes, whereas YK-3-237 exerted stimulatory effects. In summary, our data demonstrate increased oxidative stress in ASCs of aging patients, and decline of adipogenic capacity due to Sirt-1- mediated adiponectin downregulation in elderly patients.

The pro-inflammatory signature of lipopolysaccharide in spontaneous contracting embryoid bodies differentiated from mouse embryonic stem cells.

Embryonic stem (ES) cells differentiate towards all three germ layers, including cardiac cells and leukocytes, and may be therefore suitable to model inflammatory reactions in vitro. In the present study, embryoid bodies differentiated from mouse ES cells were treated with increasing doses of lipopolysaccharide (LPS) to mimic infection with gram-negative bacteria. LPS treatment dose-dependent increased contraction frequency of cardiac cell areas and calcium spikes and increased protein expression of α-actinin. LPS treatment increased the expression of the macrophage marker CD68 and CD69, which is upregulated after activation on T cells, B cells and NK cells. LPS dose-dependent increased protein expression of toll-like receptor 4 (TLR4). Moreover, upregulation of NLR family pyrin domain containing 3 (NLRP3), IL-1ß and cleaved caspase 1 was observed, indicating activation of inflammasome. In parallel, generation of reactive oxygen species (ROS), nitric oxide (NO), and expression of NOX1, NOX2, NOX4 and eNOS occurred. ROS generation, NOX2 expression and NO generation were downregulated by the TLR4 receptor antagonist TAK-242 which abolished the LPS-induced positive chronotropic effect of LPS. In conclusion, our data demonstrate that LPS induced a pro-inflammatory cellular immune response in tissues derived from ES cells, recommending the in vitro model of embryoid bodies for inflammation research.

Generation of a Syngeneic Heterozygous ACVRL1(wt/mut) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis.

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

Induction of Stem-Cell-Derived Cardiomyogenesis by Fibroblast Growth Factor 10 (FGF10) and Its Interplay with Cardiotrophin-1 (CT-1).

For heart regeneration purposes, embryonic stem cell (ES)-based strategies have been developed to induce the proliferation of cardiac progenitor cells towards cardiomyocytes. Fibroblast growth factor 10 (FGF10) contributes to cardiac development and induces cardiomyocyte differentiation in vitro. Yet, among pro-cardiogenic factors, including cardiotrophin-1 (CT-1), the hyperplastic function of FGF10 in cardiomyocyte turnover remains to be further characterized. We investigated the proliferative effects of FGF10 on ES-derived cardiac progenitor cells in the intermediate developmental stage and examined the putative interplay between FGF10 and CT-1 in cardiomyocyte proliferation. Mouse ES cells were treated with FGF10 and/or CT-1. Differential expression of cardiomyocyte-specific gene markers was analyzed at transcript and protein levels. Substantial upregulation of sarcomeric α-actinin was detected by qPCR, flow cytometry, Western blot and immunocytochemistry. FGF10 enhanced the expression of other structural proteins (MLC-2a, MLC-2v and TNNT2), transcriptional factors (NKX2-5 and GATA4), and proliferation markers (Aurora B and YAP-1). FGF10/CT-1 co-administration led to an upregulation of proliferation markers, suggesting the synergistic potential of FGF10 + CT-1 on cardiomyogenesis. In summary, we provided evidence that FGF10 and CT-1 induce cardiomyocyte structural proteins, associated transcription factors, and cardiac cell proliferation, which could be applicable in therapies to replenish damaged cardiomyocytes.

The role of ceramide accumulation in human induced pluripotent stem cell-derived cardiomyocytes on mitochondrial oxidative stress and mitophagy.

Oversupply 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 cell (hiPSC). Mature CMs derived from hiPSC 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 ß-oxidation 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. Our 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.

The nicotinamide phosphoribosyltransferase antagonist FK866 inhibits growth of prostate tumour spheroids and increases doxorubicin retention without changes in drug transporter and cancer stem cell protein expression.

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.

Zoxazolamine-induced stimulation of cardiomyogenesis from embryonic stem cells is mediated by Ca2+, nitric oxide and ATP release.

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.

Longitudinal metabolic profiling of cardiomyocytes derived from human-induced pluripotent stem cells.

Human-induced pluripotent stem cells (h-iPSCs) are a unique in vitro model for cardiovascular research. To realize the potential applications of h-iPSCs-derived cardiomyocytes (CMs) for drug testing or regenerative medicine and disease modeling, characterization of the metabolic features is critical. Here, we show the transcriptional profile during stages of cardiomyogenesis of h-iPSCs-derived CMs. CM differentiation was not only characterized by the expression of mature structural components (MLC2v, MYH7) but also accompanied by a significant increase in mature metabolic gene expression and activity. Our data revealed a distinct substrate switch from glucose to fatty acids utilization for ATP production. Basal respiration and respiratory capacity in 9 days h-iPSCs-derived CMs were glycolysis-dependent with a shift towards a more oxidative metabolic phenotype at 14 and 28 day old CMs. Furthermore, mitochondrial analysis characterized the early and mature forms of mitochondria during cardiomyogenesis. These results suggest that changes in cellular metabolic phenotype are accompanied by increased O2 consumption and ATP synthesis to fulfill the metabolic needs of mature CMs activity. To further determine functionality, the physiological response of h-iPSCs-derived CMs to ß-adrenergic stimulation was tested. These data provide a unique in vitro human heart model for the understanding of CM physiology and metabolic function which may provide useful insight into metabolic diseases as well as novel therapeutic options.

Omega-3 and Omega-6 polyunsaturated fatty acids stimulate vascular differentiation of mouse embryonic stem cells.

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.

The milk thistle (Silybum marianum) compound Silibinin stimulates leukopoiesis from mouse embryonic stem cells.

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.

Silibinin from Silybum marianum Stimulates Embryonic Stem Cell Vascular Differentiation via the STAT3/PI3-K/AKT Axis and Nitric Oxide.

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.

The Milk Thistle (Silybum marianum) Compound Silibinin Inhibits Cardiomyogenesis of Embryonic Stem Cells by Interfering with Angiotensin II 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.

Embryonic Stem Cells for Tissue Biocompatibility, Angiogenesis, and Inflammation Testing.

AIM: To introduce embryoid bodies derived from mouse embryonic stem (ES) cells, which differentiate blood vessel-like structures and leukocytes, as a novel in vitro model system for biocompatibility, inflammation, and angiogenesis studies. METHODOLOGY/RESULTS: Punched spherical discs of bioabsorbable polymers (ε-caprolactone and L-lactide in different compositions) with a diameter of 2 mm and a thickness of 0.2 mm were inoculated with embryoid bodies for cocultivation. As reference material for biocompatible, nonbioabsorbable, and bioincompatible materials, polymer punched discs of petriPERM (PP) membrane (polytetrafluoroethylene) as well as polyvinylchloride (PVC) were used. Tissue outgrowth on the polymer discs decreased and cell toxicity increased upon confrontation on bioabsorbable biomaterials and PVC. Bioabsorbable polymers as well as PVC decreased the branching points and total tube length of CD31-positive vascular structures in embryoid bodies. With the exception of PP, all applied materials increased the differentiation of CD68-positive macrophages and the generation of reactive oxygen species, which is indicative of proinflammatory processes upon contact of tissue with biomaterials. Consequently, cocultivation with polymers increased secretion of the cytokines interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α. CONCLUSION: Three-dimensional tissues cultivated from ES cells are well-suited for testing the biocompatibility, the vascular response, and the inflammatory reaction towards bioabsorbable and nonbioabsorbable polymers.

Mechanical strain stimulates vasculogenesis and expression of angiogenesis guidance molecules of embryonic stem cells through elevation of intracellular calcium, reactive oxygen species and nitric oxide generation.

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.

Differential effects of high and low strength magnetic fields on mouse embryonic development and vasculogenesis of embryonic stem cells.

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.

Impact of Arachidonic Acid and the Leukotriene Signaling Pathway on Vasculogenesis of Mouse Embryonic Stem Cells.

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.

Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells.

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.

Stimulation of vasculogenesis and leukopoiesis of embryonic stem cells by extracellular transfer RNA and ribosomal RNA.

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.

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP.

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 ß,γ-methylenadenosine 5'-triphosphate (ß,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-ß (PLC-ß) 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.

Stimulation of cardiomyogenesis from mouse embryonic stem cells by nuclear translocation of cardiotrophin-1.

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, H2DCF, 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.