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Lung Fibroblasts (lung + fibroblast)
Kinds of Lung Fibroblasts Selected AbstractsIncrease of lipid peroxidation by cisplatin in WI38 cells but not in SV40-transformed WI38 cellsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2003Hsiu-Chuan Yen Abstract Cisplatin (CPT) is an effective anticancer drug that causes cumulative toxicity to normal tissues. It has been suggested that CPT damages normal cells by causing oxidative stress, but it is not known whether it can induce similar oxidative damage to tumor cells. In this study, by using normal human lung fibroblast (W138) cells and SV40-transformed WI38 (VA13) cells as a model, we compared the effect of CPT on cytotoxicity, apoptosis, lipid peroxidation, and mitochondrial gene expression, which could be regulated by oxidative stress, between normal and tumor cells. CPT induced greater growth inhibition and percentage of apoptotic cells in VA13 cells. However, levels of esterified F2 -isoprostanes and 4-hydroxy-2-nonenal, two specific products of lipid peroxidation, were increased by CPT in WI38 cells, but not in VA13 cells. Furthermore, the transcript level of mitochondrial 12S rRNA was augmented by CPT in both cells, but to a higher degree in WI38 cells. The data suggest a correlation between lipid peroxidation and cytotoxicity or increased mitochondrial transcript levels in WI38 cells but not in VA13 cells. The results also indicate an altered response of oxidative damage and mitochondrial gene regulation to CPT in the transformed phenotype of WI38 cells. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:39,46, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10059 [source] Acute exposure of human lung cells to 1,3-butadiene diepoxide results in G1 and G2 cell cycle arrestENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2005Michael Schmiederer Abstract 1,3-butadiene (BD) causes genetic damage, including adduct formation, sister chomatid exchange, and point mutations. Previous studies have focused on the types of genetic damage and tumors found after long-term exposure of rodents to butadiene. This study examined the effect of the most active BD metabolite, butadiene diepoxide (BDO2), on cell cycle entry and progression in human lung fibroblasts (LU cells) with a normal diploid karyotype. Serum-arrested (G0) LU cells were exposed to BDO2 for 1 hr and stimulated to divide with medium containing 10% fetal bovine serum. The BDO2 -treated LU cells were evaluated for cell cycle progression, nuclear localization of arrest mediators, mitotic index, and cellular proliferation. The BDO2 -treated cells demonstrated a substantial inhibition of cell proliferation when treated with 100 ,M BDO2 for 1 hr. No appreciable levels of apoptosis or mitotic figures were observed in the BDO2 -treated cells through 96 hr posttreatment. Flow cytometric analysis revealed that the lack of proliferation in BDO2 -treated LU cells was related to G1 arrest in about half of the cells and a delayed progression through S and G2 arrest in nearly all of the remaining cells. Both G1 and G2 arrest were prolonged and only a very small percentage of BDO2 -treated cells were eventually able to replicate. Increased nuclear localization of both p53 and p21cip1 was observed in BDO2 -treated cells, suggesting that the cell cycle arrest was p21cip1 -mediated. These results demonstrate that BDO2 induces cell cycle perturbation and arrest even with short-term exposure that does not produce other pathologic cellular effects. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc. [source] Downregulation of protease-activated receptor-1 in human lung fibroblasts is specifically mediated by the prostaglandin E2 receptor EP2 through cAMP elevation and protein kinase AFEBS JOURNAL, Issue 14 2008Elena Sokolova Many cellular functions of lung fibroblasts are controlled by protease-activated receptors (PARs). In fibrotic diseases, PAR-1 plays a major role in controlling fibroproliferative and inflammatory responses. Therefore, in these diseases, regulation of PAR-1 expression plays an important role. Using the selective prostaglandin EP2 receptor agonist butaprost and cAMP-elevating agents, we show here that prostaglandin (PG)E2, via the prostanoid receptor EP2 and subsequent cAMP elevation, downregulates mRNA and protein levels of PAR-1 in human lung fibroblasts. Under these conditions, the functional response of PAR-1 in fibroblasts is reduced. These effects are specific for PGE2. Activation of other receptors coupled to cAMP elevation, such as ,-adrenergic and adenosine receptors, does not reproduce the effects of PGE2. PGE2 -mediated downregulation of PAR-1 depends mainly on protein kinase A activity, but does not depend on another cAMP effector, the exchange protein activated by cAMP. PGE2 -induced reduction of PAR-1 level is not due to a decrease of PAR-1 mRNA stability, but rather to transcriptional regulation. The present results provide further insights into the therapeutic potential of PGE2 to specifically control fibroblast function in fibrotic diseases. [source] Length-Dependent Uptake of DNA-Wrapped Single-Walled Carbon Nanotubes,ADVANCED MATERIALS, Issue 7 2007L. Becker A length threshold for cell uptake of DNA-wrapped single-walled carbon nanotubes (SWNTs) by human lung fibroblasts (IMR90) is identified. Competitive uptake experiments with well-defined and characterized length fractions show that SWNTs above the length threshold are excluded from the cell, whereas SWNTs labeled with Cy3-derivatized DNA below the threshold are able to access the cell interior, as shown in the fluorescence image and on the cover. [source] Developmental differences in the immortalization of lung fibroblasts by telomeraseAGING CELL, Issue 5 2003Nicholas R. Forsyth Summary The role of ambient (21%) and physiological oxygen (2,5%) in the immortalization of fetal vs. adult human lung fibroblasts was examined. Growth in low oxygen and antioxidants extended the lifespan of both fetal and adult strains. As the ectopic expression of telomerase could immortalize adult lung fibroblasts cultured in ambient oxygen, the lifespan-shortening effects of 21% oxygen must have been largely limited to telomeres. By contrast, fetal lung fibroblasts could not be immortalized in ambient oxygen in spite of telomere elongation by telomerase, suggesting more widespread oxidative damage. The long-term culture requirements for the immortalization of WI-38 fetal lung fibroblasts included supplementation with N-(tert) butyl hydroxylamine, dexamethasone, zinc and vitamin B12, in addition to growth in physiological oxygen. The mechanisms regulating telomere shortening remain controversial. The present results suggest that both end-replication and oxidative damage events contribute to telomere shortening in lung fibroblasts in vitro. These observations emphasize the need for better analytical techniques to distinguish whether the correlation of short telomeres with disease and mortality in humans reflects the consequences of increased proliferation, telomere shortening as a result of oxidative damage or some combination of these processes. [source] INDIVIDUAL AND COMBINED CYTOTOXIC EFFECTS OF THE MAJOR FOUR AFLATOXINS IN DIFFERENT IN VITRO STABILIZED SYSTEMSJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2010CORNELIA BRAICU ABSTRACT The present study aims to investigate the cytotoxic effect of the major aflatoxins (B1, B2, G2 and G2) and also aflatoxin combination, using a simple, rapid and cheap cytotoxicity test like MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay in three in vitro models (human umbilical vein endothelial cells [HUVEC], human lung fibroblasts [HFL] and A2780 cell line) and to extrapolate the data to in vivo situation using a prediction model. A difference in cell sensitivity has been observed for B1 and B1 + B2, in the following order A2789 > HFL > HUVEC, while for B2, G1, G2, Mix (B1 + B2 + G1 + G2) the order was HFL > A2789 > HUVEC when comparing the IC50 (half maximal inhibitory concentration) values. We confirm that in vitro cytotoxicity test MTT assay is able to predict in vivo toxicity, at least for aflatoxins using the prediction model. The values of LD50 (lethal dose 50%) calculated from experiments are different for each cell line. This fact may indicate that some species are more resistant than other and target organs are not necessarily those predicted, because the A2780 ovarian cancer cells seem to be more sensitive to B1 than cells of endothelial or fibroblasts origin. PRACTICAL APPLICATIONS This study is in concordance with the international tendency that refined the current techniques to lessen pain or distress, to reduce the number of animals necessary for a particular test or to replace animals with non-whole-animal models, such as in vitro cell cultures. The practical application of such methodologies may help solve the economic problem related to very expensive in vivo toxicology studies and implement preventive methods based on the calculated data and known mechanism of action of individual or combined toxins easily studied in vitro. The nature of coexistence of many types of mycotoxins in complex environmental samples, such as food and water, has been reported worldwide. How these mycotoxins might affect human health in combination is largely unknown. This study had, as a goal, to test the toxicity of the four aflatoxins and aflatoxin combination on human cells. Due to the lack of aflatoxins mixture data regarding the human cytotoxicity, the aim of this study was to specify, evaluate and predict the combined effects of mycotoxin mixtures. [source] Epidemiologic aspects and laboratory features of enterovirus infections in Western Germany, 2000,2005JOURNAL OF MEDICAL VIROLOGY, Issue 7 2007Bernhard Roth Abstract From 2000 to 2005, a total of 1,096 enterovirus infections were diagnosed either by isolation of virus from cell culture or by RT-PCR (5,non-coding region (NCR)). Typing of viruses (n,=,674) was carried out by immunofluorescence with monoclonal antibodies, neutralization test or molecular methods. Seasons with high enterovirus activity were characterized by high prevalence of echovirus 30 (62.2% in 2000, 25.5% in 2001) and echovirus 13 (34.5% in 2001). In contrast, in the 2003 season, which had very low enterovirus activity, these types were rare. During this season, cell culture sensitivity (human colonic carcinoma cells and human embryonic lung fibroblasts (HEL)) was exceptionally low. In order to determine the type of "non-cultivable" enteroviruses, purified RNA from selected stool samples was subjected to direct molecular typing. VP1/2A-specific fragments were amplified by RT-PCR, cloned and sequenced. The predominant virus identified was coxsackie A. Consequently, rhabdomyosarcom cells were introduced into the daily routine, which improved the isolation of enteroviruses. Echovirus 30 was again most commonly isolated during seasons 2004 and 2005 with increasing enterovirus activity. In conclusion, high prevalence of echovirus 30 and 13 is indicative of seasons with high enterovirus activity. The type of circulating enteroviruses may influence isolation of enterovirus from cell culture. RT-PCR (VP1/2A) combined with cloning and sequencing of amplicons is a useful tool for viral typing directly from stool samples. In cases of severe enterovirus infection, virological diagnosis should not solely rely on virus isolation from cell culture. J. Med. Virol. 79:956-962, 2007. © 2007 Wiley-Liss, Inc. [source] Antitumour activity and specificity as a function of substitutions in the lipophilic sector of helical lactoferrin-derived peptideJOURNAL OF PEPTIDE SCIENCE, Issue 5 2003Nannan Yang Abstract A peptide L5 (PAWRKAFRWAWRMLKKAA), derived from the N -terminal ,-helical region of bovine lactoferrin (LFB 14,31), that is highly active against several tumour cell lines was reported earlier. In this study, a number of L5 analogues were designed in order to investigate how subsequent replacements of the aromatic amino acids in L5 with three amino acids representing different structural parameters influenced antitumour activity and tumour cell specificity relative to normal human cells. The Trp residues were substituted by Lys, Ile or Ala, while the Phe residue was substituted with Ala. The resulting peptides were investigated for their activity against prokaryotic cells, four tumour cell lines, human lung fibroblasts and human erythrocytes. Most of the peptides were highly active against both E. coli and S. aureus. The peptides were more active against the tumour cell lines than against normal eukaryotic cells but the activity against normal fibroblasts varied more among the peptides than did their antitumour activities. The results revealed that aromatic residues located opposite the cationic sector in L5 were more critical for antitumour activity than were aromatic residues located adjacent to the cationic sector. The biological responses for the peptides against tumour cell lines, fibroblasts, S. aureus (but not E. coli), were highly correlated with the amino acid descriptors used in our QSAR model. The result obtained from the QSAR study identified specific structural features that were important for lytic activity and membrane specificity. Certain structural properties in positions 3, 9 and 11 were shown to be important for antitumour activity, while additional structural properties in position 7 were found to be important with respect to tumour cell specificity. This information may offer a possibility for de novo design of an antitumour peptide with an improved therapeutic index. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Gastroprotective and cytotoxic effect of semisynthetic ferruginol derivativesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 2 2007Carlos Areche The gastroprotective abietane diterpene ferruginol has been shown to present high cytotoxicity. In order to obtain active compounds with less cytotoxicity, 18 semisynthetic ferruginol derivatives and totarol were assessed for their gastroprotective effects in the HCl/ethanol-induced gastric lesion model in mice, as well as for cytotoxicity in human gastric epithelial cells (AGS) and human lung fibroblasts (MRC-5). At 20 mg kg,1, the greatest gastroprotective effects were provided by abieta-8,11,13-triene (1), abieta-8,11,13-trien-12-yl-2-chloropropanoate (8), abieta-8,11,13-trien-12-yl propenoate (9), 12-(2,3,4,6-tetra- O -acetyl-,-D-glucopyranosyloxy)-abieta-8,11,13-triene (17) and 12-(,-D-galactopyranosyloxy)-abieta-8,11,13-triene (18), all of which were as active as the reference drug lansoprazole at 20 mg kg,1, reducing gastric lesions by 69, 76, 67, 72 and 61%, respectively. No relation was observed between lipophilicity and the gastroprotective effect. Compounds that showed the greatest cytotoxicity towards AGS cells were ferruginol (2), the corresponding formate (5), acetate (6), propionate (7), 8, 9, 12-(,-D-glucopyranosyloxy)-abieta-8,11,13-triene (16), 18 and totarol (20) (IC50 18,44 ,M). Ferruginol and compounds 5,9, 16, 18 and 20 were the most toxic compounds against fibroblasts (IC50 19,56 ,M), with a correlation to AGS cells. The derivative 19 was much more active against AGS cells than towards fibroblasts. The best activity/cytotoxicity ratio was found for compound 17, with a lesion index comparable with lansoprazole at 20 mg kg,1 and cytotoxicity >1000 ,M towards MRC-5 and AGS cells, respectively. In conclusion, some derivatives showed a better gastroprotective effect/cytotoxicity ratio than the parent compound ferruginol. A total of 13 new compounds are reported here for the first time. [source] Alcohol Primes the Airway for Increased Interleukin-13 SignalingALCOHOLISM, Issue 3 2009Patrick O. Mitchell Background:, Using an experimental model of airway fibrosis following lung transplantation, we recently showed that chronic alcohol ingestion by donor rats amplifies airway fibrosis in the recipient. Associated with alcohol-mediated amplification of airway fibrosis is increased transforming growth factor ,-1(TGF,1) and ,-smooth muscle actin expression. Other studies have shown that interleukin-13 (IL-13) modulates TGF,1 signaling during experimentally-induced airway fibrosis. Therefore, we hypothesized that IL-13 is a component of alcohol-mediated amplification of pro-fibrotic mediators in the alcoholic lung. Methods:, To test this hypothesis, we analyzed tracheal epithelial cells and type II alveolar cells from control- or alcohol-fed rats, alcohol-treated mouse lung fibroblasts, and human bronchial epithelial cells in vitro for expression of various components of the IL-13 signaling pathway. Signaling via the IL-13 pathway was assessed by measuring levels of phosphorylated signal transducers and activators of transcription-6 (STAT6). In addition, we performed heterotopic tracheal transplantation using control-fed and alcohol-fed donor rats and analyzed tracheal allografts for expression of components of the IL-13 signaling pathway by RT-PCR and immunocytochemical analyses. Results:, Interleukin-13 expression was detected in type II alveolar epithelial cells and human bronchial epithelial cells, but not in lung fibroblasts. IL-13 expression was decreased in whole lung and type II cells in response to alcohol exposure. In all cell types analyzed, expression of IL-13 signaling receptor (IL-13R,1) mRNA was markedly increased. In contrast, mRNA and protein expression of the IL-13 decoy receptor (IL-13R,2) were decreased in all cells analyzed. Exposure to alcohol also increased STAT6 phosphorylation in response to IL-13 and lipopolysaccharide. Conclusions:, Data from multiple cell types in the pulmonary system suggest that IL-13 and its receptors play a role in alcohol-mediated activation of pro-fibrotic pathways. Taken together, these data suggest that alcohol primes the airway for increased IL-13 signaling and subsequent tissue remodeling upon injury such as transplantation. [source] Increased Fibronectin Expression in Lung in the Setting of Chronic Alcohol AbuseALCOHOLISM, Issue 4 2007Ellen L. Burnham Rationale: The incidence and severity of the acute respiratory distress syndrome (ARDS) is increased in individuals who abuse alcohol. One possible mechanism by which alcohol increases susceptibility to acute lung injury is through alterations in alveolar macrophage function and induction of tissue remodeling activity. Our objective was to determine whether alcohol abuse, independent of other comorbidities, alters fibronectin and metalloproteinase gene expression in alveolar macrophages and in epithelial lining fluid (ELF) of the lung. Methods: Otherwise healthy subjects with alcohol abuse (n=21) and smoking-matched controls (n=17) underwent bronchoalveolar lavage. Alveolar macrophage fibronectin and matrix metalloproteinase (MMP) mRNA expression were measured via reverse transcription-polymerase chain reaction. The supernatant from cultured alveolar macrophages and lung ELF were tested for their ability to induce fibronectin and MMP-9 gene transcription in cell-based assays. Results: Alveolar macrophages from subjects with alcohol abuse demonstrated increased fibronectin mRNA expression (p<0.001), and their ELF also elicited more fibronectin gene transcription in lung fibroblasts compared with controls (p<0.001). In contrast, alveolar macrophages from subjects with alcohol abuse had decreased MMP-9 and MMP-2 mRNA expression (p<0.03 and p<0.005, respectively). Similarly, the supernatant (p<0.001) and ELF (p<0.01) from these subjects induced less MMP-9 gene transcription in THP-1 cells. Discussion: Alcohol abuse is associated with increased fibronectin mRNA expression in alveolar macrophages and increased fibronectin-inducing activity in the ELF. This appears to be a specific effect as other tissue remodeling genes, such as MMPs, were not equally affected. These findings suggest activation of tissue remodeling that may contribute to the increased susceptibility for the ARDS observed in alcoholism. [source] Post-transcriptional regulation of plasminogen activator inhibitor-1 by intracellular iron in cultured human lung fibroblasts,interaction of an 81-kDa nuclear protein with the 3,-UTRJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 5 2005K. S. RADHA Summary., The proteinase inhibitor, type-1 plasminogen activator inhibitor (PAI-1), is a major regulator of the plasminogen activator system involved in plasmin formation and fibrinolysis. The present study explores the effects of intracellular iron on the expression of PAI-1 and associated cell-surface plasmin activity in human lung fibroblasts; and reports the presence of a novel iron-responsive protein. ELISA revealed a dose-dependent increase in PAI-1 antigen levels expressed in the conditioned medium of cells treated with deferoxamine, in the three cell lines studied. A concomitant increase in mRNA levels was also observed by Northern analyses. Presaturation with ferric citrate quenched the effect of deferoxamine. Experiments with transcription and translation inhibitors on TIG 3-20 cells demonstrated that intracellular iron modulated PAI-1 expression at the post-transcriptional level with the requirement of de-novo protein synthesis. Electrophoretic mobility shift assay and UV crosslinking assays revealed the presence of an ,,81-kDa nuclear protein that interacted with the 3,-UTR of PAI-1 mRNA in an iron-sensitive manner. Finally, we demonstrated that the increased PAI-1 is functional in suppressing cell-surface plasmin activity, a process that can affect wound healing and tissue remodeling. [source] Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2005M. V. OVANESOV Summary., Exposure of tissue factor (TF)-bearing cells to blood is the initial event in coagulation and intravascular thrombus formation. However, the mechanisms which determine thrombus growth remain poorly understood. To explore whether the procoagulant activity of vessel wall-bound cells regulates thrombus expansion, we studied in vitro spatial clot growth initiated by cultured human cells of different types in contact pathway-inhibited, non-flowing human plasma. Human aortic endothelial cells, smooth muscle cells, macrophages and lung fibroblasts differed in their ability to support thrombin generation in microplate assay with peaks of generated thrombin of 60 ± 53 nmol L,1, 135 ± 57 nmol L,1, 218 ± 55 nmol L,1 and 407 ± 59 nmol L,1 (mean ± SD), respectively. Real-time videomicroscopy revealed the initiation and spatial growth phases of clot formation. Different procoagulant activity of cell monolayers was manifested as up to 4-fold difference in the lag times of clot formation. In contrast, the clot growth rate, which characterized propagation of clotting from the cell surface to plasma, was largely independent of cell type (, 30% difference). Experiments with factor VII (FVII)-, FVIII-, FX- or FXI-deficient plasmas and annexin V revealed that (i) cell surface-associated extrinsic Xase was critical for initiation of clotting; (ii) intrinsic Xase regulated only the growth phase; and (iii) the contribution of plasma phospholipid surfaces in the growth phase was predominant. We conclude that the role of TF-bearing initiator cells is limited to the initial stage of clot formation. The functioning of intrinsic Xase in plasma provides the primary mechanism of sustained and far-ranging propagation of coagulation leading to the physical expansion of a fibrin clot. [source] Effects of dexamethasone on proliferation, chemotaxis, collagen I, and fibronectin-metabolism of human fetal lung fibroblastsPEDIATRIC PULMONOLOGY, Issue 1 2001R.E. Brenner MD Abstract Premature infants at risk for bronchopulmonary dysplasia (BPD) are often treated with dexamethasone (Dex), which has been shown to suppress inflammatory processes in the lung. To elucidate a possible direct influence on the fibroproliferative component of the disease, we studied the effects of Dex in therapeutic and supratherapeutic dosages (5,50 nmol/L) on proliferation, chemotaxis, procollagen I, and fibronectin metabolism of human fetal lung fibroblasts in vitro. Proliferation was inhibited by Dex in a dose-dependent manner. Chemotactic activity in response to conditioned medium of human fetal fibroblasts also showed a dose-dependent inhibition after pretreatment with Dex. The amount of procollagen I C-terminal propeptide and fibronectin per cell in the cell culturesupernatant was increased in the presence of Dex. Our results show that Dex does not uniformly suppress the fibroproliferative activity of human fetal lung fibroblasts, which may explain in part the unsatisfactory long-term effects of Dex treatment in BPD. Pediatr Pulmonol. 2001; 32:1,7. © 2001 Wiley-Liss, Inc. [source] Reference gene selection for real-time polymerase chain reaction in human lung cells subjected to cyclic mechanical strainRESPIROLOGY, Issue 7 2008Liao PINHU Background and objective: The respiratory system is constantly exposed to mechanical forces that influence cellular phenotype in health and disease. Quantitative real-time PCR (qPCR) is widely used to determine gene expression. The validity of qPCR depends on using stable reference genes for normalization. The effect of cyclic mechanical strain on reference gene expression by lung epithelial, fibroblast and endothelial cells has not been studied systematically. Methods: The stability of expression of fourteen potential reference genes in response to six different regimens of cyclic mechanical strain was ranked using the geNorm tool in human lung epithelial cell lines (A549 and H441), human fetal lung fibroblasts (HFL-1), human lung microvascular endothelial cells, primary human lung fibroblasts and primary human alveolar type 2 (hAT2) cells. The expression variation of these reference genes was also screened in unstimulated whole human lung. Results: The stability of the selected reference genes varied within and between cell types, the variation in expression being greatest in primary cultures of hAT2. Correspondingly, the effect of expressing message for the stretch responsive gene IL-8 normalized to the 14 reference genes was greatest in the hAT2 cells, there being an almost fivefold difference in mRNA relative change comparing different reference genes in the same samples. The minimum number of genes required to derive a reliable normalization factor for experiments on single lung cell types undergoing mechanical strain was two and for whole human lung it was four. Conclusions: These results demonstrate that the optimal reference genes for lung cells subjected to CMS are cell type specific. [source] Dabigatran, a direct thrombin inhibitor, demonstrates antifibrotic effects on lung fibroblastsARTHRITIS & RHEUMATISM, Issue 11 2009Galina S. Bogatkevich Objective Myofibroblasts are the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of lung parenchyma associated with pulmonary fibrosis. We previously reported that thrombin activates protease-activated receptor 1 (PAR-1) and induces a myofibroblast phenotype in normal lung fibroblasts resembling the phenotype of scleroderma lung myofibroblasts. We undertook this study to investigate whether a selective direct thrombin inhibitor, dabigatran, interferes with signal transduction in human lung fibroblasts induced by thrombin and mediated via PAR-1. Methods Lung fibroblast proliferation was analyzed using the Quick Cell Proliferation Assay. Expression and organization of ,-smooth muscle actin (,-SMA) was studied by immunofluorescence staining and immunoblotting. Contractile activity of lung fibroblasts was measured by a collagen gel contraction assay. Connective tissue growth factor (CTGF) and type I collagen expression was analyzed on Western blots. Results Dabigatran, at concentrations of 50,1,000 ng/ml, inhibited thrombin-induced cell proliferation, ,-SMA expression and organization, and the production of collagen and CTGF in normal lung fibroblasts. Moreover, when treated with dabigatran (1 ,g/ml), scleroderma lung myofibroblasts produced 6-fold less ,-SMA, 3-fold less CTGF, and 2-fold less type I collagen compared with untreated cells. Conclusion Dabigatran restrains important profibrotic events in lung fibroblasts and warrants study as a potential antifibrotic drug for the treatment of fibrosing lung diseases such as scleroderma lung disease and idiopathic pulmonary fibrosis. [source] Pivotal role of connective tissue growth factor in lung fibrosis: MAPK-dependent transcriptional activation of type I collagenARTHRITIS & RHEUMATISM, Issue 7 2009Markella Ponticos Objective Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor , (TGF,) and is a mediator of some profibrotic effects of TGF, in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I ,2) in this mouse model and in human pulmonary fibroblasts. Methods Transgenic mice that were carrying luciferase and ,-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts. Results In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by ,25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin-treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling. Conclusion Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Col1a2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc. [source] Role of hypoxia and cAMP in the transdifferentiation of human fetal cardiac fibroblasts: Implications for progression to scarring in autoimmune-associated congenital heart blockARTHRITIS & RHEUMATISM, Issue 12 2007Robert M. Clancy Objective Identification of isolated congenital heart block (CHB) predicts, with near certainty, the presence of maternal anti-SSA/Ro antibodies; however, the 2% incidence of CHB in first offspring of anti-SSA/Ro+ mothers, 20% recurrence in subsequent pregnancies, and discordance in identical twins suggest that an environmental factor amplifies the effect of the antibody. Accordingly, this study was carried out to explore the hypothesis that hypoxia potentiates a profibrosing phenotype of the fetal cardiac fibroblast. Methods Evidence of an effect of hypoxia was sought by immunohistologic evaluation of CHB-affected fetal heart tissue and by determination of erythropoietin levels in cord blood. The in vitro effect of hypoxia on gene expression and phenotype in fibroblasts derived from fetal hearts and lungs was investigated by Affymetrix arrays, quantitative polymerase chain reaction (PCR), immunofluorescence, and immunoblotting. Results In vivo hypoxic exposure was supported by the prominent intracellular fibroblast expression of hypoxia-inducible factor 1, in conduction tissue from 2 fetuses in whom CHB led to death. The possibility that hypoxia was sustained was suggested by significantly elevated erythropoietin levels in cord blood from CHB-affected, as compared with unaffected, anti-SSA/Ro,exposed neonates. In vitro exposure of cardiac fibroblasts to hypoxia resulted in transdifferentiation to myofibroblasts (a scarring phenotype), as demonstrated on immunoblots and immunofluorescence by increased expression of smooth muscle actin (SMA), an effect not seen in lung fibroblasts. Hypoxia-exposed cardiac fibroblasts expressed adrenomedullin at 4-fold increased levels, as determined by Affymetrix array, quantitative PCR, and immunofluorescence, thus focusing attention on cAMP as a modulator of fibrosis. MDL12,330A, an adenylate cyclase inhibitor that lowers the levels of cAMP, increased expression of fibrosis-related proteins (mammalian target of rapamycin, SMA, plasminogen activator inhibitor type 1, and type I collagen), while the cAMP activator forskolin attenuated transforming growth factor ,,elicited fibrosing end points in the cardiac fibroblasts. Conclusion These findings provide evidence that hypoxia may amplify the injurious effects of anti-SSA/Ro antibodies. Modulation of cAMP may be a key component in the scarring phenotype. Further assessment of the susceptibility of cardiac fibroblasts to cAMP modulation offers a new research direction in CHB. [source] Attenuation of Bleomycin-Induced Lung Fibrosis by Oxymatrine Is Associated with Regulation of Fibroblast Proliferation and Collagen Production in Primary CultureBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2008Xiaohong Chen Oxymatrine is an alkaloid extracted from the Chinese herb Sophora japonica (Sophora flavescens Ait.) with capacities of anti-inflammation, inhibition of immune reaction, antivirus, protection against acute lung injury and antihepatic fibrosis. In this study, the effect of oxymatrine on pulmonary fibrosis was investigated using a bleomycin-induced pulmonary fibrosis mouse model. The results showed that bleomycin challenge provoked severe pulmonary fibrosis with marked increase in hydroxyproline content of lung tissue and lung fibrosis fraction, which was prevented by oxymatrine in a dose-dependent manner. In addition, bleomycin injection resulted in a marked increase of myeloperoxidase activity and malondialdehyde level that was attenuated by oxymatrine. Administration of oxymatrine inhibited the proliferation of murine lung fibroblasts, arrested the cells at G0/G1 phase and reduced the expression of cell cycle regulatory protein, cyclin D1 in vitro. Furthermore, the steady-state production of collagen and the expression of ,1(I) pro-collagen and ,2(I) pro-collagen mRNA in fibroblasts were inhibited by oxymatrine in a dose-dependent manner. These results suggested that oxymatrine may attenuate pulmonary fibrosis induced by bleomycin in mice, partly through inhibition of inflammatory response and lipid peroxidation in lung induced by bleomycin and reduction of fibroblast proliferation and collagen synthesis. [source] Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditionsBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2004Carla Alexandra Da Silva Stem cell factor (SCF) is a major growth factor for mast cells, promoting their differentiation and chemotaxis. Its expression is regulated by glucocorticoids in inflammatory conditions, showing an early increased protein expression, before the expected anti-inflammatory decrease (Da Silva et al., Br. J. Pharmacol. 2002:135,1634). We here evaluated the early kinetic of SCF expression regulated by interleukin (IL)-1,, budesonide and the combination of both in human lung fibroblasts in culture. Budesonide potentiated the IL-1, -enhanced expression of SCF mRNA (+103%) and protein (+98%) very shortly after treatment (at 30 min and 1 h, respectively). A gentle downregulation followed. This potentiating effect of budesonide was related to increased SCF mRNA stability and SCF gene transcription. Deletion of a ,B-like site that we identified in the first intron of the SCF gene, in a luciferase reporter system, abolished the potentiation by budesonide, as well as the effect of IL-1, alone, as compared to the wild-type construction activity. All budesonide-induced effects were glucocorticoid-receptor dependent, since they were reproduced by dexamethasone and blocked by RU486. IL-1,+budesonide did not affect the relative expression of the soluble and membrane-bound forms of SCF. In conclusion, our results clearly show that glucocorticoids act very early to adversely increase the expression of SCF mRNA and protein in the inflammatory conditions created by IL-1,, and that this effect involves increased mRNA stability and increased gene expression through activation of the NF- ,B-like responsive element. British Journal of Pharmacology (2004) 141, 75,84. doi:10.1038/sj.bjp.0705598 [source] Interleukin-6 receptor superantagonist Sant7 inhibits TGF-,-induced proliferation of human lung fibroblastsCELL PROLIFERATION, Issue 3 2008L. Gallelli Therefore, the aim of this study was to investigate in primary cultures of normal and fibrotic human lung fibroblasts (HLF), exposed to either IL-6 or TGF-,1, the effects on phosphorylation of mitogen-activated protein kinases (MAPK) and cell growth of IL-6 signalling inhibition, performed by the IL-6 receptor superantagonist Sant7.Materials and methods:,MAPK phosphorylation was detected by Western blotting, HLF viability and proliferation were evaluated using the trypan blue staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Results:,Sant7, at a concentration of 1 µg/mL, was capable of significantly inhibiting HLF proliferation and MAPK phosphorylation induced by cell exposure to IL-6 (100 ng/mL) or TGF-,1 (10 ng/mL), whose actions were more evident in fibrotic cells. Conclusions:,These findings suggest that, in HLFs derived from patients with ILDs, the proliferative mechanisms activated by TGF-,1 are at least in part mediated by an increased release of IL-6, leading to phosphorylation-dependent MAPK activation. Such preliminary findings may thus open new therapeutic perspectives for fibrogenic ILDs, based on inhibition of signal transduction pathways stimulated by the IL-6 receptor. [source] Platelet-activating factor stimulates ovine foetal pulmonary vascular smooth muscle cell proliferation: role of nuclear factor-kappa B and cyclin-dependent kinasesCELL PROLIFERATION, Issue 2 2008B. O. Ibe Objective: Platelet-activating factor (PAF) is implicated in pathogenesis of persistent pulmonary hypertension of the neonate (PPHN); PAF is a mitogen for lung fibroblasts. PAF's role in pulmonary vascular smooth muscle cell (PVSMC) proliferation and in hypoxia-induced pulmonary vein (PV) remodelling has not been established and mechanisms for PAF's cell-proliferative effects are not well understood. We investigated involvement of PAF and PAF receptors in PVSMC proliferation. Materials and methods: Cells from pulmonary arteries (SMC-PA) and veins (SMC-PV) were serum starved for 72 h in 5% CO2 in air (normoxia). They were cultured for 24 h more in normoxia or 2% O2 (hypoxia) in 0.1% or 10% foetal bovine serum with 5 µCi/well of [3H]-thymidine, with and without 10 nm PAF. Nuclear factor-kappa B (NF-,B), CDK2 and CDK4 protein expression, and their roles in cell proliferation control were studied. Results: PAF and hypoxia increased SMC-PA and SMC-PV proliferation. WEB2170 inhibited PAF-induced cell proliferation while lyso-PAF had no effect. SMC-PV proliferated more than SMC-PA and PAF plus hypoxia augmented NF-,B protein expression. NF-,B inhibitory peptide attenuated PAF-induced cell proliferation by 50% and PAF increased CDK2 and CDK4 protein expression. The data show that hypoxia and PAF up-regulate PVSMC proliferation via PAF receptor-specific pathway involving NF-,B, CDK2 and CDK4 activations. Conclusion: They suggest that in vivo, in foetal lung low-oxygen environment, where PAF level is high, proliferation of PVSMC will occur readily to modulate PV development and that failure of down-regulation of PAF effects postnatally may result in PPHN. [source] Opposite effect of fluticasone and salmeterol on fibronectin and tenascin-C expression in primary human lung fibroblastsCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2009M. Degen Summary Background Airway remodelling is a key feature of asthma and chronic obstructive pulmonary disease (COPD). The remodelling process involves the deposition of extracellular matrix (ECM) proteins within the airways. Current therapies for asthma and COPD consist of inhaled corticosteroids and long-acting ,2 -agonists (LABA). However, their effect on airway remodelling is not well understood so far. Objective In this study we investigated the effect of fluticasone and salmeterol, either alone or in combination, on fibronectin and tenascin-C protein, isoform, and mRNA levels in primary human lung fibroblasts. Methods In our model, fibroblasts cultured in serum-free medium represented a non-inflammatory condition and stimulation with 5% fetal calf serum and/or TGF-,1 mimicked a pro-fibrotic environment with activation of tissue repair. Using these two different conditions, the effects of fluticasone and salmeterol on fibronectin and tenascin-C protein and mRNA levels were analysed by immunoblotting and semi-quantitative RT-PCR. Results In both conditions, fluticasone increased fibronectin transcript and protein levels, whereas it decreased those of tenascin-C. Salmeterol neither affected fibronectin and tenascin-C synthesis significantly nor did it influence the effect of fluticasone when applied in combination. Furthermore, we found that treatment with fluticasone had an opposite effect on extra domain A and B containing fibronectin isoforms generated by alternative splicing compared with total fibronectin transcript levels, whereas tenascin-C isoforms were not differently modulated by fluticasone. Conclusions Our results indicate that standard therapies for inflammatory lung disorders influence ECM protein composition and relative expression levels. In contrast to corticosteroids, LABA did not significantly alter the expression of tenascin-C and fibronectin in cultures of primary human lung fibroblasts. [source] Airway epithelium-derived transforming growth factor-, is a regulator of fibroblast proliferation in both fibrotic and normal subjectsCLINICAL & EXPERIMENTAL ALLERGY, Issue 8 2008K. E. Hostettler Summary Background In the healthy lung, airway epithelial cells (AEC) regulate fibroblast proliferation through release of soluble factors, such as prostaglandins and proteins. Fibroproliferative diseases and airway remodelling may result from an inadequate generation of suppressive factors by AEC or the inability of fibroblasts to respond to them appropriately. Objective The aim of this study was to study the effect of primary human AEC on the proliferation of fibroblasts obtained from healthy and fibrotic lungs in an interactive cell culture model. Results Conditioned medium (CM) from 14 out of 16 AEC lines significantly inhibited proliferation of normal human lung fibroblasts by 51.2±6.0%. The proliferation of fibroblasts derived from patients with lung fibrosis was equally inhibited by CM of AEC. The inhibitory effect of AEC-CM was completely reversed when fibroblasts were pre-incubated with 2.5 ,m indomethacin. Furthermore, primary human AEC, but not fibroblasts, secrete TGF-,, and the inhibitory effect of the AEC-CM was blocked by neutralizing anti-TGF-, antibodies. Conclusion These results demonstrate that AEC actively inhibit the proliferation of both normal and fibrotic fibroblasts via TGF-,, which induces the prostaglandin E2 synthesis in fibroblasts. The data indicate that proliferative lung diseases may be treated using the epithelial cell as the target of medication. [source] Interleukin-13 acts as an apoptotic effector on lung epithelial cells and induces pro-fibrotic gene expression in lung fibroblastsCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2008A. Borowski Summary Background IL-13 promotes acute allergic asthma and is discussed to play a role in late asthmatic features such as fibrotic processes and airway remodelling. The contributions of IL-13-mediated mechanisms to subepithelial events related to fibrosis are not yet settled. Objective We investigated the impact of IL-13 on lung epithelial cells as apoptotic effector and on lung fibroblasts as inducer of pro-fibrotic gene expression. Methods Using the two lung epithelial cell lines A549 and BEAS-2B as well as primary lung epithelial cells, we investigated the capability of IL-13 to induce apoptosis by both flow-cytometry and ELISA. The ability of IL-13 to increase the expression of pro-fibrotic genes and to exert influence on the expression of its own receptor was investigated by real-time quantitative PCR measurement of mRNAs encoding collagen I, collagen III, basic fibroblast growth factor (bFGF), ,-smooth muscle actin (,-SMA) and the IL-13 receptor ,1 (IL-13R,1) chain in human primary lung fibroblasts. The specificity of IL-13-mediated cellular responses was confirmed by means of an inhibitory monoclonal antibody directed to the IL-13 receptor. Results IL-13 induces apoptosis in lung epithelial cell lines as well as in primary lung epithelial cells. Furthermore, IL-13 increases the expression of mRNA for ,-SMA and collagen III, but not for bFGF in human primary lung fibroblasts. The susceptibility of lung fibroblasts to IL-13-induced up-regulation of pro-fibrotic genes is associated with the regulation of IL-13 receptor expression. IL-13-dependent fibrosis-associated effects could be inhibited by antibody-mediated blockade of the IL-13R,1 subunit. Conclusion Our findings indicate a function of IL-13 as a mediator in fibrotic processes leading to loss of functional airway tissue in asthma. They also highlight the therapeutic potential of specifically targeting the interaction between IL-13 and its receptor. [source] Induction of eotaxin production by interleukin-4, interleukin-13 and lipopolysaccharide by nasal fibroblastsCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2004M. Nonaka Summary Background There is growing evidence that eotaxin is a key mediator in the development of tissue eosinophilia. Fibroblasts are a major source of eotaxin. The severity of diseases with eosinophilic inflammation like nasal polyposis, atopic dermatitis and asthma, where Th2-type cytokines (IL-4 and IL-13) and TGF-, are expressed locally, was shown to correlate with bacterial factors such as lipopolysaccharide (LPS) rather than allergen. Objective We examined eotaxin production by nasal fibroblasts stimulated with IL-4 or IL-13 alone or in combination with LPS, and the effect of TGF-,1 on it. Moreover, we compared the magnitude of eotaxin produced by nasal fibroblasts with that produced by lung or skin fibroblasts. Methods Fibroblast lines were established from human biopsy tissue. The expression of eotaxin mRNA was evaluated by RT-PCR. The amount of eotaxin in the supernatants was measured by ELISA. Results IL-4, but not IL-13, synergized with LPS to produce eotaxin in a dose- and time-dependent manner. Sequential treatment of nasal fibroblasts with IL-4 and LPS did not have any effect. But when IL-4 and LPS were added together, synergy for eotaxin production was observed. Moreover, this synergy was observed in nasal and skin fibroblasts, but not in lung fibroblasts. The production of eotaxin by IL-4 and LPS was modulated by TGF-,1. Conclusion These results suggest that a co-stimulus like LPS is necessary for IL-4 to make a strong induction of eotaxin in eosinophilic inflammations such as nasal polyposis. Modulation by TGF-,1 may have important implications for the development of eosinophilic inflammation. [source] | ||||||||||||||||||||||||||||