54 resultados para Fibroblasts
Resumo:
Glucocorticoids (GC) represent the most commonly used drugs for the treatment of acute and chronic inflammatory skin diseases. However, the topical long-term therapy of GC is limited by the occurrence of skin atrophy. Most interestingly, although GC inhibit proliferation of human fibroblasts, they exert a pronounced anti-apoptopic action. In the present study, we further elucidated the molecular mechanism of the GC dexamethasone (Dex) to protect human fibroblasts from programmed cell death. Dex not only significantly alters the expression of the cytosolic isoenzyme sphingosine kinase 1 but also initiated an enhanced intracellular formation of the sphingolipid sphingosine 1-phosphate (S1P). Investigations using S1P (3) ((-/-)) -fibroblasts revealed that this S1P-receptor subtype is essential for the Dex-induced cytoprotection. Moreover, we demonstrate that the ATP-binding cassette (ABC)-transporter ABCC1 is upregulated by Dex and may represent a crucial carrier to transport S1P from the cytosol to the S1P(3)-receptor subtype.
Resumo:
The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.
Resumo:
Mechanical forces are essential for connective tissue homeostasis. The extracellular matrix (ECM) plays a key role in the transmission of forces generated by the organism (e.g. muscle contraction) and externally applied (e.g. gravity). The expression of specific ECM proteins such as collagens and tenascin-C, as well as of matrix metalloproteinases, involved in their turnover, is influenced by mechanical stimuli. The precise mechanisms by which mechanical strains are translated into chemical signals and lead to differential gene expression are however not fully understood. Cell-matrix adhesion sites are good candidates for hosting a "mechanosensory switch", as they transmit forces from the ECM to the cytoskeleton and vice versa by physically linking the cytoskeleton to the ECM. Integrins, transmembrane proteins located to these adhesion sites, have been shown to trigger a set of internal signaling cascades after mechanical stimulation. We have shown that the expression level of tenascin-C directly correlates with externally applied mechanical stress, as well as with RhoA/RhoA-dependent kinase-mediated cytoskeletal tension. Presumably other genes are regulated in a similar manner. The changes in ECM composition and mechanical properties derived from mechanical stress are relevant in medical intervention after ligament and tendon injury.
Resumo:
Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond to cyclic strain by a varying degree of focal adhesion assembly and actin reorganization. We speculated that the individual shape of the cells, which is linked to cytoskeletal structure and pre-stress, might tune these integrin-dependent mechanotransduction events. To this aim, fibronectin circles, squares and rectangles of identical surface area (2000μm(2)) were micro-contact printed onto elastomeric substrates. Fibroblasts plated on these patterns occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to patterned cells for 30min, and changes in cytoskeleton and cell-matrix adhesions were quantified after fluorescence staining. After strain, megakaryocytic leukemia-1 protein translocated to the nucleus in most cells, indicating efficient RhoA activation independently of cell shape. However, circular and square cells (with radial symmetry) showed a significantly greater increase in the number of actin stress fibers and vinculin-positive focal adhesions after cyclic strain than rectangular (bipolar) cells of identical size. Conversely, cyclic strain induced larger changes in pY397-FAK positive focal complexes and zyxin relocation from focal adhesions to stress fibers in bipolar compared to symmetric cells. Thus, radially symmetric cells responded to cyclic strain with a larger increase in assembly, whereas bipolar cells reacted with more pronounced reorganization of actin stress fibers and matrix contacts. We conclude that integrin-mediated responses to external mechanical strain are differentially modulated in cells that have the same spreading area but different geometries, and do not only depend on mere cell size.
Resumo:
OBJECTIVE Catecholamines released from β-adrenergic neurons upon stress can interfere with periodontal regeneration. The cellular mechanisms, however, are unclear. Here, we assessed the effect of catecholamines on proliferation of periodontal fibroblasts. METHODS Fibroblasts from the gingiva and the periodontal ligament were exposed to agonists of the β-adrenergic receptors; isoproterenol (ISO, non-selective β-adrenergic agonist), salbutamol (SAL, selective β2-adrenergic receptor agonist) and BRL 37344 (BRL selective β3-receptor agonist). Proliferation was stimulated with platelet-derived growth factor-BB (PDGF-BB). Pharmacological inhibitors and gene expression analysis further revealed β-adrenergic signalling. RESULTS Gingiva and periodontal ligament fibroblast express the β2-adrenergic receptor. ISO and SAL but not BRL decreased proliferation of fibroblasts in the presence of PDGF-BB. The inhibitory effect of β-adrenergic signalling on proliferation but not protein synthesis in response to PDGF-BB was reduced by propranolol, a non-selective β-adrenergic antagonist. CONCLUSIONS These results suggest that β2-receptor agonists can reduce the mitogenic response of periodontal fibroblasts. These data add to the compelling concept that blocking of β2-receptor signalling can support tissue maintenance and regeneration.
Resumo:
Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy metabolism and oncogene-induced premature senescence. The S phase of the cell cycle, polyploidy events, DNA double strand breaks (DSBs) and DNA single strand breaks (SSBs) were increased in BPV-1-positive cells compared to control fibroblasts. BPV-1-mediated oxidative stress may contribute to telomere dysfunction in sarcoid-derived fibroblasts. Loss of mitochondrial membrane potential and concurrent elevation in intracellular ATP production may be a consequence of changes in energy-supplying pathways in BPV-1-positive cells which is also typical for cancer cells. Shifts in energy metabolism may support rapid proliferation in cells infected by BPV-1. Nevertheless, sarcoid-derived fibroblasts representing a heterogeneous cell fraction vary in some aspects of metabolic phenotype due to a dual role of BPV-1 in cell transformation and oncogene-induced premature senescence. This was shown with increased senescence-associated β-galactosidase (SA-β-gal) activity. Taken together, metabolic phenotypes in sarcoid-derived fibroblasts are plastic, which are similar to greater plasticity of cancer tissues than normal tissues.
Resumo:
Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10 μg/mL VALE and 1 μg/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo.
Resumo:
Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro.
Resumo:
BACKGROUND The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium. METHODS Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction. RESULTS The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1-stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed. CONCLUSION L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.
Resumo:
Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1β and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.
Resumo:
BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.
Resumo:
Background/Aims: Ceramide kinase (CerK) catalyzes the generation of the sphingolipid ceramide-1-phosphate (C1P) which regulates various cellular functions including cell growth and death, and inflammation. Here, we used a novel catalytic inhibitor of CerK, NVP-231, and CerK knockout cells to investigate the contribution of CerK to proliferation and inflammation in renal mesangial cells and fibroblasts. Methods: Cells were treated with NVP-231 and [3H]-thymidine incorporation into DNA, [3H]-arachidonic acid release, prostaglandin E2 (PGE2) synthesis, cell cycle distribution, and apoptosis were determined. Results: Treatment of rat mesangial cells and mouse renal fibroblasts with NVP-231 decreased DNA synthesis, but not of agonist-stimulated arachidonic acid release or PGE2 synthesis. Similarly, proliferation but not arachidonic acid release or PGE2 synthesis was reduced in CERK knockout renal fibroblasts. The anti-proliferative effect of NVP-231 on mesangial cells was due to M phase arrest as determined using the mitosis markers phospho-histone H3, cdc2 and polo-like kinase-1, and induction of apoptosis. Moreover, loss of CerK sensitized cells towards stress-induced apoptosis. Conclusions: Our data demonstrate that CerK induces proliferation but not PGE2 formation of renal mesangial cells and fibroblasts, and suggest that targeted CerK inhibition has potential for treating mesangioproliferative kidney diseases.
Resumo:
In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.
Resumo:
It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.