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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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Safoura Mazrouei reports a relationship with Uniklinik Jena that includes: non-financial support and travel reimbursement., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Free fatty acids (FFA), hyperglycemia, and inflammatory cytokines are major mediators of β-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.

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., (© 2020 The Authors. Clinical and Experimental Pharmacology and Physiology published by John Wiley & Sons Australia, Ltd.)

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., (© 2020 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.)

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 Ser 1177 under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr 495 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© 2018 John Wiley & Sons, Ltd.)

The milk thistle ( Silybum marianum (L.) Gaertn.) compound silibinin may be an inhibitor of the angiotensin II type 1 (AT 1 ) 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 (Ca 2+ ) spiking. In contrast, angiotensin II (Ang II) treatment stimulated cardiomyogenesis as well as contraction and Ca 2+ spiking frequency, which were abolished in the presence of silibinin. Intracellular Ca 2+ 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 AT 1 receptor. Ang II treatment activated extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH 2 -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 AT 1 receptor.

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., Competing Interests: The authors declare there are no competing interests. Antonia Mantakaki is an employee of Teleflex Incorporated.

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., Competing Interests: All authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this study., (Copyright © 2018, Spandidos Publications.)

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 N ω -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., Competing Interests: The authors declare no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

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.

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., (© 2017 S. Karger AG, Basel.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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., (© 2016 S. Karger AG, Basel.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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

Background: Cardiotrophin-1 (CT-1) controls cardiomyogenesis of mouse embryonic stem (ES) cells., Objectives: To investigate the signaling pathway underlying the action of CT-1 on cardiac cell differentiation., Methods: Protein expression was analyzed by western blot technique and cardiac areas by immunohistochemistry. Calcium, reactive oxygen species (ROS) and nitric oxide (NO) were assessed by microfluorometry using fluo-4, 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., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

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

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.

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.

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.

Aims: Intermedin (IMD) is a novel member of the calcitonin gene-related peptide family, which acts via calcitonin receptor-like receptors (CLRs), mediating activation of cAMP signalling. The main objective of the present study was to analyse the molecular mechanisms of the differential effects of IMD on the macromolecule permeability of endothelial cells of different vascular beds., Methods and Results: Here we demonstrate that IMD increases permeability of rat coronary microvascular endothelial cells (RCECs) and reduces permeability of human umbilical vein endothelial cells (HUVECs) and rat aortic endothelial cells via CLRs and cAMP. Intermedin causes a derangement of the actin cytoskeleton accompanied by loss of vascular endothelial cadherin (VE-cadherin) in RCECs, while it causes a rearrangement of the actin cytoskeleton and VE-cadherin at cell-cell junctions in HUVECs. Intermedin inactivates the RhoA/Rho-kinase (Rock) pathway in both cell types; however, it inactivates Rac1 in RCECs but not in HUVECs. Inhibition and rescue experiments demonstrate that both RhoA and Rac1 are required for the RCEC barrier stability, while in HUVECs the inhibition of RhoA/Rock signalling does not interfere with basal permeability., Conclusion: The opposite effects of IMD on permeability of RCECs and HUVECs are due to differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover, Rac1 activity is regulated by the RhoA/Rock pathway in RCECs but not in HUVECs.

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

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

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