Rat liver myofibroblasts (MFB) are the key cells involved in the deposition of extracellular matrix in fibrotic liver. They were isolated by repeated passaging of non-parenchymal cell fraction and cultured in 3-dimensional (3D) collagen gel mimicking tissue. The transfer of MFB from plastic dishes to collagen resulted in the change in their shape from large and spread to slender with long extensions. The expression of transforming growth factor-beta1 (TGF-beta1) and of MFB markers, alpha-smooth muscle actin (alpha-SMA) and cellular fibronectin (EDA-FN), on protein level was significantly decreased in collagen gel. The gel did not change the expression of metalloproteinase MMP-2 but activated the proenzyme. The experiments with inhibitors of metabolic pathways showed that EDA-FN and alpha-SMA were differently regulated. The expression of EDA-FN required functional TGF-beta1 receptors and was also dependent on the activity of protein kinases MEK1 and MEK2. alpha-SMA expression was primarily determined by the 3D environment. Fibroblast growth factor-1 (FGF-1) in combination with heparin decreased the expression of alpha-SMA and increased the expression of EDA-FN in the cells on plastic. The cellular environment may influence the cells per se and may modify the action of other agents.

• MeSH
• aktiny metabolismus   MeSH
• benzamidy MeSH
• biologické markery metabolismus   MeSH
• butadieny MeSH
• dioxoly MeSH
• fibroblastový růstový faktor 1 metabolismus   MeSH
• fibronektiny metabolismus   MeSH
• játra cytologie   MeSH
• kultivační techniky * MeSH
• kultivované buňky MeSH
• matrixová metaloproteinasa 2 metabolismus   MeSH
• myofibroblasty cytologie   metabolismus   MeSH
• nitrily MeSH
• potkani Sprague-Dawley MeSH
• transformující růstový faktor beta metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide MeSH Prohlížeč
• Acta2 protein, rat MeSH Prohlížeč
• aktiny MeSH
• benzamidy MeSH
• biologické markery MeSH
• butadieny MeSH
• dioxoly MeSH
• fibroblastový růstový faktor 1 MeSH
• fibronektiny MeSH
• matrixová metaloproteinasa 2 MeSH
• Mmp2 protein, rat MeSH Prohlížeč
• nitrily MeSH
• transformující růstový faktor beta MeSH
• U 0126 MeSH Prohlížeč

Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial‑mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period.

• MeSH
• aktiny metabolismus   MeSH
• buněčná diferenciace MeSH
• crista neuralis cytologie   MeSH
• dárci tkání * MeSH
• dospělí MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• fenotyp MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fibronektiny biosyntéza   MeSH
• imunohistochemie MeSH
• keratinocyty cytologie   metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• kokultivační techniky MeSH
• lidé MeSH
• mezoderm cytologie   MeSH
• myofibroblasty cytologie   MeSH
• nestin metabolismus   MeSH
• neuroplasticita MeSH
• novorozenec MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• stanovení celkové genové exprese MeSH
• stárnutí fyziologie   MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ACTA2 protein, human MeSH Prohlížeč
• aktiny MeSH
• fibronektiny MeSH
• nestin MeSH

Atrial natriuretic peptide antifibrotic properties are mainly described in cardiac myocytes or in induced cardiac myofibroblasts (Angiotensin II or TGF-beta induced differentiation). In the present work, we investigate the effects of ANP/NPRA/cGMP system in modulating rat cardiac fibroblasts function. Cardiac fibroblasts were isolated from adult Wistar male rats and cultured in the presence of serum in order to induce fibroblasts differentiation. Cultures were then treated with ANP (1 microM), 8-Br-cGMP (100 microM) or IBMX (100 microM), a non-specific phosphodiesterases inhibitor. ANP significantly decreased proliferation rate and collagen secretion. Its effect was mimicked by the cGMP analog, while combining ANP with 8-Br-cGMP did not lead to additional effects. Moreover intracellular cGMP levels were elevated when cells were incubated with ANP confirming that ANP intracellular pathway is mediated by cGMP. Additionally, immunoblotting and immunofluorescence were used to confirm the presence of guanylyl cyclase specific natriuretic peptide receptors A and B. Finally we scanned specific cGMP dependent PDEs via RT-qPCR, and noticed that inhibiting all PDEs led to an important decrease in proliferation rate. Effect of ANP became more prominent after 10 culture days, confirming the importance of ANP in fibroblasts to myofibroblasts differentiation. Uncovering cellular aspects of ANP/NPRA/cGMP signaling system provided more elements to help understand cardiac fibrotic process.

• MeSH
• atriální natriuretický faktor aplikace a dávkování   MeSH
• buněčná diferenciace účinky léků   fyziologie   MeSH
• fibroblasty cytologie   účinky léků   MeSH
• kardiomyocyty cytologie   účinky léků   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• myofibroblasty cytologie   účinky léků   MeSH
• potkani Wistar MeSH
• srdeční komory cytologie   účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• atriální natriuretický faktor MeSH

Fibrotic diseases are a group of pathologies with high incidence and mortality. Despite extensive research efforts, effective therapies are still not available. Understanding the molecular mechanisms driving the onset, progression and possible resolution of fibrosis is a prerequisite to the development of successful therapies. The central role of the TGF-β pathway and myofibroblasts in the pathogenesis of fibrosis is now generally accepted. The possible mechanisms of myofibroblast elimination or dedifferentiation, on the other hand, are still almost uncharted territory. Here we show that sustained expression of some components of MAPK signaling pathway (PDGFB, Ha-Ras(G12V) or the transcription factor EGR4) in primary chicken embryo dermal myofibroblasts results in a loss of autocrine TGF-β signaling and suppression of the myofibroblastic phenotype, characterized by the loss of alpha smooth muscle actin fibers and a substantial reduction in the production of extracellular matrix. Detailed analysis of the possible molecular mechanisms employed by EGR4 revealed FOXG1, BAMBI, NAB1, NAB2 and DUSP5 genes forming an EGR4 regulated network counteracting autocrine TGF-β signaling. We have also found that a combination of chemical inhibition of TGF-β signaling and perturbation of MAPK signaling with phorbol ester mimics the anti-fibrotic effects of PDGFB, Ha-Ras(G12V) and EGR4.

• Klíčová slova
• EGR4, FOXG1, Ha-Ras(G12V), Microarrays, Myofibroblast, PDGFB, TGF-β,
• MeSH
• aktiny genetika   metabolismus   MeSH
• dediferenciace buněk * MeSH
• forbolové estery farmakologie   MeSH
• kuřecí embryo MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• myofibroblasty cytologie   metabolismus   MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• forbolové estery MeSH
• mitogenem aktivované proteinkinasy MeSH
• transformující růstový faktor beta MeSH

We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-β1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.

• MeSH
• aktiny metabolismus   MeSH
• antigen CD146 metabolismus   MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčný rodokmen účinky léků   MeSH
• chondrogeneze účinky léků   MeSH
• endoteliální buňky cytologie   účinky léků   metabolismus   MeSH
• imunohistochemie MeSH
• kmenové buňky účinky léků   patologie   MeSH
• kultivační média bez séra farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• molár třetí patologie   MeSH
• myocyty hladké svaloviny cytologie   účinky léků   metabolismus   MeSH
• myofibroblasty cytologie   účinky léků   metabolismus   MeSH
• neurony cytologie   účinky léků   metabolismus   MeSH
• osteogeneze účinky léků   MeSH
• průtoková cytometrie MeSH
• zaklíněný zub patologie   MeSH
• zubní dřeň patologie   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• antigen CD146 MeSH
• kultivační média bez séra MeSH

Cardiac fibroblast-myofibroblast transformation (CMT) is a critical event in the initiation of myocardial fibrosis. Notch signaling has been shown to regulate myofibroblast transformation from other kinds of cells. However, whether Notch signaling is also involved in CMT remains unclear. In the present study, expressions of Notch receptors in cardiac fibroblasts (CFs) were examined, effects of Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and transforming growth factor-beta1 (TGF-beta1) on CMT were determined by increasing alpha-smooth muscle actin (alpha-SMA) expression and collagen synthesis, and Notch signaling was examined by analyzing expressions of Notch receptors. The results showed that: (1) Notch receptor 1, 2, 3 and 4 were all expressed in CFs; (2) DAPT promoted CMT in a time-dependent manner; (3) During the period of CMT induced by TGF-beta1, expressions of Notch receptor 1, 3 and 4 in CFs were down-regulated, whereas there was no change for Notch receptor 2. Moreover, the downtrends of Notch 1, 3 and 4 were corresponding to the trend growth of alpha-SMA expression and collagen synthesis. These results suggested that inhibiting of Notch signaling might promote CMT. The down-regulations of Notch receptor 1, 3 and 4 induced by TGF-beta1 may facilitate CMT. In conclusion, inhibition of Notch signaling might be a novel mechanism of CMT in myocardial fibrosis.

• MeSH
• buněčná diferenciace MeSH
• down regulace MeSH
• fibroblasty cytologie   metabolismus   MeSH
• kardiomyocyty cytologie   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• myofibroblasty cytologie   metabolismus   MeSH
• novorozená zvířata MeSH
• potkani Sprague-Dawley MeSH
• receptory Notch metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory Notch MeSH