All-trans-retinoic acid (atRA) is a potent ligand that regulates gene expression and is used to treat several skin disorders. Hyaluronic acid (HA) was previously conjugated with atRA (HA-atRA) to obtain a novel amphiphilic compound. HA-atRA forms micelles that incorporate hydrophobic molecules and facilitate their transport through the skin. The aim of this study was to determine the influence of HA-atRA on gene expression in skin cells and to compare it with that of unbound atRA. Gene expression was investigated using microarrays and a luciferase system with a canonical atRA promoter. HA-atRA upregulated gene expression similarly to atRA. However, HA-atRA activated the expression of cholesterol metabolism genes, unlike atRA. Further investigation using HPLC and filipin III staining suggested that the treated cells induced cholesterol synthesis to replenish the cholesterol removed from the cells by HA-atRA. HA modified with oleate (HA-C18:1) removed cholesterol from the cells similarly to HA-atRA, suggesting that the cholesterol removal stemmed from the amphiphilic nature of the two derivatives. HA-atRA induces retinoid signaling. Thus, HA-atRA could be used to treat skin diseases, such as acne and psoriasis, where the combined action of atRA signaling and anti-inflammatory cholesterol removal may be potentially beneficial.

• Klíčová slova
• Delcore, HyRetin, amphiphilic hyaluronan, cholesterol, hyaluronic acid, nanocarrier, retinoic acid,
• MeSH
• cholesterol metabolismus   MeSH
• exprese genu MeSH
• kyselina hyaluronová farmakologie   MeSH
• retinoidy * farmakologie   MeSH
• tretinoin * farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• kyselina hyaluronová MeSH
• retinoidy * MeSH
• tretinoin * MeSH

An innovative multi-step phase separation process was used to prepare tissue culture for the polystyrene-based, hierarchically structured substrates, which mimicked in vivo microenvironment and architecture. Macro- (pore area from 3000 to 18,000 µm2; roughness (Ra) 7.2 ± 0.1 µm) and meso- (pore area from 50 to 300 µm2; Ra 1.1 ± 0.1 µm) structured substrates covered with micro-pores (area around 3 µm2) were prepared and characterised. Both types of substrate were suitable for human-induced pluripotent stem cell (hiPSC) cultivation and were found to be beneficial for the induction of cardiomyogenesis in hiPSC. This was confirmed both by the number of promoted proliferated cells and the expressions of specific markers (Nkx2.5, MYH6, MYL2, and MYL7). Moreover, the substrates amplified the fluorescence signal when Ca2+ flow was monitored. This property, together with cytocompatibility, make this material especially suitable for in vitro studies of cell/material interactions within tissue-mimicking environments.

• Klíčová slova
• biomimetic, cardiomyogenesis, fluorescence signal, human-induced pluripotent stem cells, surfaces,
• MeSH
• biokompatibilní materiály chemie   MeSH
• buněčná diferenciace * MeSH
• fluorescence * MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• kardiomyocyty cytologie   MeSH
• lidé MeSH
• polystyreny chemie   MeSH
• proliferace buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biokompatibilní materiály MeSH
• polystyreny MeSH

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

• Klíčová slova
• biocompatibility, conducting polymer, neurogenesis, polypyrrole, stem cells,
• MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• buněčné linie MeSH
• embryoidní tělíska cytologie   účinky léků   MeSH
• exprese genu účinky léků   MeSH
• molekulární struktura MeSH
• myší embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• myši MeSH
• nervové kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• neurogeneze účinky léků   genetika   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• polymery chemie   farmakologie   MeSH
• pyrroly chemie   farmakologie   MeSH
• transkripční faktor PAX6 genetika   MeSH
• transkripční faktory bHLH genetika   MeSH
• transkripční faktory SOXB1 genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Ascl1 protein, mouse MeSH Prohlížeč
• polymery MeSH
• polypyrrole MeSH Prohlížeč
• pyrroly MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory bHLH MeSH
• transkripční faktory SOXB1 MeSH

The differentiation of pluripotent embryonic stem (ES) cells into various lineages in vitro represents an important tool for studying the mechanisms underlying mammalian embryogenesis. It is a key technique in studies evaluating the molecular mechanisms of cardiomyogenesis and heart development and also in embryotoxicology. Herein, modest modifications of the basic protocol for ES cell differentiation into cardiomyocytes were evaluated in order to increase the yield and differentiation status of developed cardiomyocytes. Primarily, the data show that ES cell cultivation in the form of non-adherent embryoid bodies (EBs) for 5 days compared to 8 days significantly improved cardiomyogenic differentiation. This is illustrated by the appearance of beating foci in the adherent EBs layer at earlier phases of differentiation from day 10 up to day 16 and by the significantly higher expression of genes characteristic of cardiomyogenic differentiation (sarcomeric alpha actinin, myosin heavy chain alpha and beta, myosin light chain 2 and 7, and transcriptional factor Nkx2.5) in EBs cultivated under non-adherent conditions for 5 days. The ratio of cardiomyocytes per other cells was also potentiated in EBs cultivated in non-adherent conditions for only 5 days followed by cultivation in adherent serum-free culture conditions. Nevertheless, the alteration in the percentage of beating foci among these two tested cultivation conditions vanished at later phases and also did not affect the total number of cardiomyocytes determined as myosin heavy chain positive cells at the end of the differentiation process on day 20. Thus, although these modifications of the conditions of ES cells differentiation may intensify cardiomyocyte differentiation, the final count of cardiomyocytes might not change. Thus, serum depletion was identified as a key factor that intensified cardiomyogenesis. Further, the treatment of EBs with N-acetylcysteine, a reactive oxygen species scavenger, did not affect the observed increase in cardiomyogenesis under serum depleted conditions. Interestingly, a mild induction of the ventricular-like phenotype of cardiomyocytes was observed in 5-day-old EBs compared to 8-day-old EBs. Overall, these findings bring crucial information on the mechanisms of ES cells differentiation into cardiomyocytes and on the establishment of efficient protocols for the cardiomyogenic differentiation of ES cells. Further, the importance of determining the absolute number of formed cardiomyocyte-like cells per seeded pluripotent cells in contrast to the simple quantification of the ratios of cells is highlighted.

• MeSH
• acetylcystein aplikace a dávkování   MeSH
• aktinin genetika   MeSH
• embryonální kmenové buňky cytologie   MeSH
• homeoboxový protein Nkx-2.5 genetika   MeSH
• kardiomyocyty cytologie   MeSH
• kultivační média bez séra * MeSH
• kultivované buňky MeSH
• myosiny genetika   MeSH
• myši MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcystein MeSH
• aktinin MeSH
• homeoboxový protein Nkx-2.5 MeSH
• kultivační média bez séra * MeSH
• myosiny MeSH
• Nkx2-5 protein, mouse MeSH Prohlížeč

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

• MeSH
• acetofenony farmakologie   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• myší embryonální kmenové buňky účinky léků   metabolismus   MeSH
• myši MeSH
• NADPH-oxidasy genetika   metabolismus   MeSH
• oniové sloučeniny farmakologie   MeSH
• oxidační stres účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny Wnt metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• synergismus léků MeSH
• transkripční faktor STAT3 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetofenony MeSH
• acetovanillone MeSH Prohlížeč
• diphenyleneiodonium MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy MeSH
• fosfatidylinositol-3-kinasy MeSH
• NADPH-oxidasy MeSH
• oniové sloučeniny MeSH
• proteiny Wnt MeSH
• protoonkogenní proteiny c-akt MeSH
• reaktivní formy kyslíku MeSH
• transkripční faktor STAT3 MeSH

Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O(2)(∙-) formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O(2)(∙-) and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O(2)(∙-) soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O(2) (∙-) formation in inflammatory macrophages.

• Klíčová slova
• L-arginine, Macrophages, NO., inducible nitric oxide synthase, superoxide anion,
• MeSH
• aktivace enzymů MeSH
• arginin imunologie   MeSH
• biopteriny analogy a deriváty   metabolismus   MeSH
• buněčné linie MeSH
• časové faktory MeSH
• Escherichia coli imunologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• lipopolysacharidy škodlivé účinky   MeSH
• makrofágy účinky léků   imunologie   metabolismus   MeSH
• myši MeSH
• NADPH-oxidasy metabolismus   MeSH
• NG-nitroargininmethylester farmakologie   MeSH
• oxid dusnatý metabolismus   MeSH
• respirační vzplanutí MeSH
• superoxidy metabolismus   MeSH
• synthasa oxidu dusnatého, typ II antagonisté a inhibitory   metabolismus   MeSH
• tyrosin analogy a deriváty   metabolismus   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-nitrotyrosine MeSH Prohlížeč
• arginin MeSH
• biopteriny MeSH
• inhibitory enzymů MeSH
• lipopolysacharidy MeSH
• NADPH-oxidasy MeSH
• NG-nitroargininmethylester MeSH
• oxid dusnatý MeSH
• sapropterin MeSH Prohlížeč
• superoxidy MeSH
• synthasa oxidu dusnatého, typ II MeSH
• tyrosin MeSH