RESUMO
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.
Assuntos
Células-Tronco Pluripotentes Induzidas , Telangiectasia Hemorrágica Hereditária , Humanos , Mutação , Células Endoteliais , Qualidade de Vida , Telangiectasia Hemorrágica Hereditária/genética , Fator de Crescimento Transformador beta/genética , Linhagem Celular , Receptores de Activinas Tipo II/genéticaRESUMO
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.
Assuntos
Citocinas , Nicotinamida Fosforribosiltransferase , Transporte Biológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos , PiperidinasRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Coração/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Proteína Homeobox Nkx-2.5/genética , Humanos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Cadeias Pesadas de Miosina/genética , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Troponina I/genética , Zoxazolamina/farmacologiaRESUMO
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.
Assuntos
Leucopoese/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Silibina/farmacologia , Silybum marianum/química , Animais , Cromonas/farmacologia , Camundongos , Morfolinas/farmacologia , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fator de Transcrição STAT3/efeitos dos fármacos , Silybum marianum/química , Silimarina/farmacologia , Animais , Cromonas/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Camundongos , Morfolinas/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , SilibinaRESUMO
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.
Assuntos
Células-Tronco Embrionárias/metabolismo , Imuno-Histoquímica/métodos , Neovascularização Patológica/metabolismo , Materiais Biocompatíveis , Diferenciação Celular , Citocinas , Células-Tronco Embrionárias/citologia , Humanos , InflamaçãoRESUMO
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.
Assuntos
Cálcio/metabolismo , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Becaplermina , Benzoxazóis/farmacologia , Fenômenos Biomecânicos , Cálcio/agonistas , Diferenciação Celular , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Embrião de Mamíferos , Corpos Embrioides/citologia , Corpos Embrioides/efeitos dos fármacos , Efrina-B2/antagonistas & inibidores , Efrina-B2/genética , Efrina-B2/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/genética , Neuropilina-1/metabolismo , Óxido Nítrico/agonistas , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/agonistas , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Semaforinas/genéticaRESUMO
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.
Assuntos
Desenvolvimento Embrionário , Campos Magnéticos , Neovascularização Fisiológica , Animais , Linhagem Celular , Estatura Cabeça-Cóccix , Perda do Embrião , Embrião de Mamíferos , Corpos Embrioides/metabolismo , Olho/embriologia , Olho/metabolismo , Feminino , Fígado/embriologia , Fígado/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Embrionárias Murinas/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aumento de PesoRESUMO
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.
Assuntos
Ácido Araquidônico/farmacologia , Leucotrienos/farmacologia , Células-Tronco Embrionárias Murinas/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Animais , Vias Biossintéticas/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Leucotrienos/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco Embrionárias Murinas/enzimologia , NADH NADPH Oxirredutases/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Diferenciação Celular/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , Neovascularização Fisiológica/genética , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Células-Tronco Embrionárias/patologia , Leucopoese/genética , Neovascularização Fisiológica/genética , RNA Ribossômico/genética , RNA de Transferência/genética , Animais , Western Blotting , Diferenciação Celular , Movimento Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Peróxido de Hidrogênio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas Imunoenzimáticas , Camundongos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxidos/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Trifosfato de Adenosina/antagonistas & inibidores , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Contração Miocárdica/efeitos dos fármacos , Gravidez , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2X/biossíntese , Receptores Purinérgicos P2X/efeitos dos fármacos , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Difosfato de Uridina/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
The ß-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 ß-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific ß-adrenergic receptor (ß-adrenoceptor) antagonist Propranolol, the ß1-adrenoceptor-specific antagonist Atenolol and the ß2-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three ß-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, ß-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 ß-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 ß-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that ß-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Antagonistas Adrenérgicos beta/administração & dosagem , Animais , Atenolol/administração & dosagem , Atenolol/farmacologia , Biomarcadores/metabolismo , Corpos Embrioides/citologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Propanolaminas/administração & dosagem , Propanolaminas/farmacologia , Propranolol/administração & dosagem , Propranolol/farmacologia , Receptores Adrenérgicos beta/metabolismo , S-Nitroso-N-Acetilpenicilamina/administração & dosagem , S-Nitroso-N-Acetilpenicilamina/farmacologia , Fatores de TempoRESUMO
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.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Endotélio Vascular/citologia , Leptina/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Gordura Abdominal/citologia , Idoso , Apoptose , Biomarcadores/metabolismo , Hipóxia Celular , Células Cultivadas , Humanos , Pessoa de Meia-Idade , Neovascularização Fisiológica , TranscriptomaRESUMO
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.
Assuntos
Corpos Embrioides/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Ativação Transcricional , alfa-Macroglobulinas/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos da Linhagem 129 , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
Embryonic stem (ES) cells are a powerful model for the development of cells responsible for the cellular immune response. Therefore, we analyzed the defense and phagocytic capacity of embryoid bodies (EBs) derived from ES cells using in the vitro inflammatory conditions caused by Escherichia coli. Further, we used this phagocytic activity to purify activated immune cells. Our data show that spontaneously differentiated 18-day-old EBs of the cell line CGR8 contained immune cells, which were positive for CD45, CD68, CD11b, F4/80, and CD19. Exposure of these EBs to E. coli with defined infection doses of bacterial colony-forming units (CFUs) led to a significant time-dependent reduction of CFUs, indicating the immune responses exerted by EBs. This was paralleled by an upregulation of inflammatory cytokines, that is, IL-1ß and TNF-α. Western blot analysis of infected EBs indicated an upregulation of CD14 and cytochrome b-245 heavy chain (NOX2). Silencing of NOX2 significantly reduced the antibacterial capacity of EBs, which was partially explained by reduction of F4/80-positive cells. To identify, isolate, and further cultivate phagocytic active cells from differentiated EBs, a cocultivation assay of differentiated ES cells with green fluorescent protein (GFP)-labeled E. coli was established. Colocalization of GFP-labeled E. coli with cells positive for CD45, CD68, and F4/80 revealed time-dependent phagocytotic uptake, which was underlined by colocalization with the LysoTracker-Red(®) dye as well as preincubation with cytochalasin D. In conclusion, a primitive immune response with efficient phagocytosis was responsible for the antibacterial capacity of differentiated EBs.
Assuntos
Corpos Embrioides/imunologia , Escherichia coli/crescimento & desenvolvimento , Fagocitose/imunologia , Aminas , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Contagem de Colônia Microbiana , Citocalasina D , Corpos Embrioides/citologia , Escherichia coli/imunologia , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologiaRESUMO
The growth of cancer cells is limited by energy supply which is regulated by the energy sensor AMP-kinase (AMPK). Hence, mimicking a low energy state may inhibit cancer growth and may be exploited in anticancer therapies. In the present study, the impact of AMPK activation on cell growth and apoptosis of DU-145 prostate cancer cells was investigated. Incubation with the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR) dose-dependently inhibited cell growth, activated AMPK, and inhibited mTOR. Furthermore, AICAR treatment activated c-Jun N-terminal kinase (JNK) and caspase-3, thereby initiating apoptosis. Within 60 min of treatment AICAR raised intracellular reactive oxygen species (ROS) which could be abolished in the presence of the free radical scavenger N-(2-mercaptopropionyl)glycin (NMPG), the AMPK inhibitor compound C (Comp C) and the respiratory chain complex I inhibitor rotenone, but not by the NADPH oxidase inhibitor VAS2870. Inhibition of ROS generation abolished AMPK activation by AICAR as well as JNK and caspase-3 activation. Furthermore, AMPK activation, JNK phosphorylation and cleaved caspase-3 upon AICAR treatment were abolished in the presence of Comp C. In summary, our data demonstrate that activation of AMPK by AICAR induces apoptosis of prostate cancer cells by a signaling pathway involving ROS, activation of JNK and cleaved caspase-3.
Assuntos
Adenilato Quinase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias da Próstata/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeos/farmacologia , Aminoimidazol Carboxamida/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Fosforilação , Serina-Treonina Quinases TOR/metabolismoAssuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Canais de Potássio Cálcio-Ativados/fisiologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Benzimidazóis/farmacologia , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Proliferação de Células , Forma Celular , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Corpos Embrioides , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Canais de Potássio , Canais de Potássio Cálcio-Ativados/agonistas , Canais de Potássio Cálcio-Ativados/metabolismo , Transcrição GênicaRESUMO
VEGF-, phosphoinositide 3-kinase (PI3K)- and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1⺠cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110α and p110δ using short hairpin RNA (shRNA), or inhibition of p110α with compound 15e and of p110δ with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110ß, and shRNA knockdown of p110ß were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCα/ßII) and rottlerin (targeting PKCδ) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCα/ßII and PKCδ but not PKCζ. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositide-dependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110α and p110δ are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Corpos Embrioides/citologia , Desenvolvimento Muscular , Miocárdio/citologia , Neovascularização Fisiológica , Proteína Quinase C/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Domínio Catalítico , Diferenciação Celular , Linhagem Celular , Cromonas/farmacologia , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Corpos Embrioides/efeitos dos fármacos , Ativação Enzimática , Camundongos , Morfolinas/farmacologia , Miocárdio/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de SinaisRESUMO
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.