Pathophysiological correlation of arginase-1 in development of type 2 diabetes from obesity in adolescents.

BACKGROUND: There is great interest to understand causal pathophysiological correlation between obesity and diabetes mellitus (DM). Vascular endothelial dysfunction is crucially involved in pathogenesis of vascular complications in DM. Recently, increased arginase expression and activity have been described as underlying mechanisms of endothelial dysfunction in DM and vascular inflammation in obesity. By limiting L-arginine bioavailability to endothelial nitric oxide synthase (NOS III), nitric oxide production is potentially impaired. METHODS: We investigated the impact of plasma from diabetic and obese adolescents on arginase and NOS III expression in cultured human endothelial cells (ECs). A total of 148 male adolescents participated in this study including 18 obese, 28 type 1-, 28 type 2-DM patients, and 74 age-matched healthy volunteers. RESULTS: A concurrent increase in arginase-1 (1.97-fold) and decrease in NOS III expression (1.45-fold) was observed in ECs exposed to type 2 diabetic plasma compared to control subjects. ECs incubated with type 1 DM plasma had a diminished NOS III level without impact on arginase-1 expression. Urea-assay featured an increased arginase activity in treated ECs with type 1- or 2-DM plasma. Despite increased pro-inflammatory cytokines and chemokines in obese plasma, arginase-1 expression/activity did not change in treated ECs. However, NOS III expression was significantly reduced. Pearson analysis revealed positive correlation between arginase-1, but not NOS III, expression with FBS in ECs treated with type 2-DM plasma. CONCLUSIONS: Our data demonstrate that increased arginase-1 expression/activity in ECs, as critical pathogenic factor is correlated with development of obesity-related type 2-DM and linked vascular disease.

Loss and Recovery of Glutaredoxin 5 Is Inducible by Diet in a Murine Model of Diabesity and Mediated by Free Fatty Acids In Vitro.

Free fatty acids (FFA), hyperglycemia, and inflammatory cytokines are major mediators of ß-cell toxicity in type 2 diabetes mellitus, impairing mitochondrial metabolism. Glutaredoxin 5 (Glrx5) is a mitochondrial protein involved in the assembly of iron-sulfur clusters required for complexes of the respiratory chain. We have provided evidence that islet cells are deprived of Glrx5, correlating with impaired insulin secretion during diabetes in genetically obese mice. In this study, we induced diabesity in C57BL/6J mice in vivo by feeding the mice a high-fat diet (HFD) and modelled the diabetic metabolism in MIN6 cells through exposure to FFA, glucose, or inflammatory cytokines in vitro. qRT-PCR, ELISA, immunohisto-/cytochemistry, bioluminescence, and respirometry were employed to study Glrx5, insulin secretion, and mitochondrial biomarkers. The HFD induced a depletion of islet Glrx5 concomitant with an obese phenotype, elevated FFA in serum and reactive oxygen species in islets, and impaired glucose tolerance. Exposure of MIN6 cells to FFA led to a loss of Glrx5 in vitro. The FFA-induced depletion of Glrx5 coincided with significantly altered mitochondrial biomarkers. In summary, we provide evidence that Glrx5 is regulated by FFA in type 2 diabetes mellitus and is linked to mitochondrial dysfunction and blunted insulin secretion.

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.

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.

Regulation of MAP kinase-mediated endothelial dysfunction in hyperglycemia via arginase I and eNOS dysregulation.

Emerging evidence suggests that arginase contributes to endothelial dysfunction in diabetes. Intracellular signaling pathways, which interplay between arginase and eNOS enzyme activity leading to the development of endothelial dysfunction in hyperglycemia are not fully understood. Here, we analyzed the possible involvement of hyperglycemia (HG) induced arginase expression in eNOS protein regulation and activity and also the impact of arginase inhibition on eNOS activity. Furthermore, the roles of p38 MAPK and Erk1/2 phosphorylation in upregulation of arginase expression and eNOS dysregulation in endothelial cells (ECs) under hyperglycemia were evaluated. Protein analysis showed a concurrent increase in arginase I expression and decrease in eNOS expression and phosphorylation at Ser1177 under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr495 in HG. Arginase inhibition prevented increased arginase activity, restored impaired NO bioavailability and reduced superoxide anion generation. Inhibition of MAP-kinases demonstrated that, unlike Erk1/2, p38 MAPK is an upstream activator in a signaling cascade leading to increased arginase I in HG conditions. P38 MAPK protein expression and phosphorylation were increased in response to HG. In the presence of a p38 MAPK inhibitor, HG-induced arginase expression was blunted. Although Erk1/2 was activated in HG, increased arginase expression was not blocked by co-treatment with an Erk1/2 inhibitor. Activation of both, p38 MAPK and Erk1/2 in HG, induced a downregulation in eNOS activity. Hence, applying MAPK inhibitors increased eNOS phosphorylation in HG. In conclusion, these findings demonstrate contributions of arginase I in the development of endothelial cell dysfunction under HG conditions via impaired eNOS regulation, which maybe mediated by p38 MAPK.

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.

Erratum: Mitochondrial G8292A and C8794T mutations in patients with Niemann-Pick disease type C.

[This corrects the article DOI: 10.3892/br.2018.1095.].

Recent advances and challenges on application of tissue engineering for treatment of congenital heart disease.

Congenital heart disease (CHD) affects a considerable number of children and adults worldwide. This implicates not only developmental disorders, high mortality, and reduced quality of life but also, high costs for the healthcare systems. CHD refers to a variety of heart and vascular malformations which could be very challenging to reconstruct the malformed region surgically, especially when the patient is an infant or a child. Advanced technology and research have offered a better mechanistic insight on the impact of CHD in the heart and vascular system of infants, children, and adults and identified potential therapeutic solutions. Many artificial materials and devices have been used for cardiovascular surgery. Surgeons and the medical industry created and evolved the ball valves to the carbon-based leaflet valves and introduced bioprosthesis as an alternative. However, with research further progressing, contracting tissue has been developed in laboratories and tissue engineering (TE) could represent a revolutionary answer for CHD surgery. Development of engineered tissue for cardiac and aortic reconstruction for developing bodies of infants and children can be very challenging. Nevertheless, using acellular scaffolds, allograft, xenografts, and autografts is already very common. Seeding of cells on surface and within scaffold is a key challenging factor for use of the above. The use of different types of stem cells has been investigated and proven to be suitable for tissue engineering. They are the most promising source of cells for heart reconstruction in a developing body, even for adults. Some stem cell types are more effective than others, with some disadvantages which may be eliminated in the future.

Mitochondrial G8292A and C8794T mutations in patients with Niemann-Pick disease type C.

Niemann-Pick disease type C (NP-C) is a neurovisceral lipid storage disorder. At the cellular level, the disorder is characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system. NP-C is transmitted in an autosomal recessive manner and is caused by mutations in either the NPC1 (95% of families) or NPC2 gene. The estimated disease incidence is 1 in 120,000 live births, but this likely represents an underestimate, as the disease may be under-diagnosed due to its highly heterogeneous presentation. Variants of adenosine triphosphatase (ATPase) subunit 6 and ATPase subunit 8 (ATPase6/8) in mitochondrial DNA (mtDNA) have been reported in different types of genetic diseases including NP-C. In the present study, the blood samples of 22 Iranian patients with NP-C and 150 healthy subjects as a control group were analyzed. The DNA of the blood samples was extracted by the salting out method and analyzed for ATPase6/8 mutations using polymerase chain reaction sequencing. Sequence variations in mitochondrial genome samples were determined via the Mitomap database. Analysis of sequencing data confirmed the existence of 11 different single nucleotide polymorphisms (SNPs) in patients with NP-C1. One of the most prevalent polymorphisms was the A8860G variant, which was observed in both affected and non-affected groups and determined to have no significant association with NP-C incidence. Amongst the 11 polymorphisms, only one was identified in the ATPase8 gene, while 9 including A8860G were observed in the ATPase6 gene. Furthermore, two SNPs, G8292A and C8792A, located in the non-coding region of mtDNA and the ATPase6 gene, respectively, exhibited significantly higher prevalence rates in NP-C1 patients compared with the control group (P<0.01). The present study suggests that there may be an association between mitochondrial ATPase6/8 mutations and the incidence of NP-C disease. In addition, the mitochondrial SNPs identified maybe pathogenic mutations involved in the development and prevalence of NP-C. Furthermore, these results suggest a higher occurrence of mutations in ATPase6 than in ATPase8 in NP-C patients.

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.

Differential expression of islet glutaredoxin 1 and 5 with high reactive oxygen species production in a mouse model of diabesity.

The onset and progression of diabetes mellitus type 2 is highly contingent on the amount of functional beta-cell mass. An underlying cause of beta-cell decay in diabetes is oxidative stress, which markedly affects the insulin producing pancreatic cells due to their poor antioxidant defence capacity. Consequently, disturbances of cellular redox signaling have been implicated to play a major role in beta-cell loss in diabetes mellitus type 2. There is evidence suggesting that the glutaredoxin (Grx) system exerts a protective role for pancreatic islets, but the exact mechanisms have not yet been elucidated. In this study, a mouse model for diabetes mellitus type 2 was used to gain further insight into the significance of Grx for the islets of Langerhans in the diabetic metabolism. We have observed distinct differences in the expression levels of Grx in pancreatic islets between obese, diabetic db mice and lean, non-diabetic controls. This finding is the first report about a decrease of Grx expression levels in pancreatic islets of diabetic mice which was accompanied by declining insulin secretion, increase of reactive oxygen species (ROS) production level, and cell cycle alterations. These data demonstrate the essential role of the Grx system for the beta-cell during metabolic stress which may provide a new target for diabetes mellitus type 2 treatment.

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.

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.

Stem Cell Spheroid-Based Sprout Assay in Three-Dimensional Fibrin Scaffold: A Novel In Vitro Model for the Study of Angiogenesis.

Angiogenesis is a complex process of critical importance during development and in physiological and pathophysiological conditions. There is considerable research interest in studying the angiogenesis cascade and consequently a need for a physiologically valid, quantitative, and cost-effective assay. In this chapter, we describe the stem cell spheroid-based sprout assay in three-dimensional fibrin scaffold which allows fast and easy screening of pro- and anti-angiogenic effects of substances with a high degree of reproducibility.

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.