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Fibroblasts Isolated (fibroblast + isolated)
Selected AbstractsPivotal 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] Effect of parathyroid hormone-related protein on fibroblast proliferation and collagen metabolism in human skinEXPERIMENTAL DERMATOLOGY, Issue 4 2002Emanuela Maioli Abstract: The parathyroid hormone-related protein (PTHrp), structurally similar to the parathyroid hormone (PTH) in its NH2 -terminal part, was first identified as a tumour-derived peptide responsible for a paraneoplastic syndrome known as humoral hypercalcemia of malignancy. The PTHrp gene is expressed not only in cancer but also in normal tissues during adult and/or fetal life, where it plays predominantly paracrine and/or autocrine roles. In the skin PTHrp produced by keratinocytes acts on fibroblasts by complex cooperative circuits involving cytokines and growth factors. In this report, we studied the direct effects of synthetic PTHrp 1,40 on proliferation and collagen synthesis and matrix metalloproteinase-2 (MMP-2) activity in cultures of fibroblasts isolated from normal human skin. Fibroblasts exposure to varying doses of PTHrp for 48 h, significantly and dose-dependently inhibited proliferation evaluated by [3H]-thymidine incorporation into DNA. A dose-dependent stimulation of cAMP released into the medium was concomitantly observed. In contrast, PTHrp had no effect on collagen synthesis evaluated either by [3H]-proline incorporation or by radioimmunoassay (RIA) of the carboxyterminal fragment of type I procollagen (PICP). MMP-2 activity, evaluated by quantitative zymographic analysis, was significantly increased by PTHrp treatment at doses of 160 and 320 nM. These findings indicate that PTHrp may play a role in normal dermal physiology by controlling both fibroblast proliferation and extracellular matrix degradation. [source] Effect of triclosan on interferon-, production and major histocompatibility complex class II expression in human gingival fibroblastsJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 10 2000Manal Mustafa Abstract Background, aims: The effect of triclosan (2,4,4,-trichloro-2,-hydroxyl-diphenyl ether) on the production of interferon-, (IFN-,) and the expression of major histocompatibility complex (MHC) class II antigen was studied in human gingival fibroblasts isolated from 4 individuals. Methods/Results: AII cell lines demonstrated high IFN-, production in 24-h cultures of human gingival fibroblasts stimulated by phytohemagglutinin (PHA) (5 ,g/ml). Human gingival fibroblasts showed a high expression of MHC class II when stimulated with 500 and 1000 pg/ml rIFN-, in 7-day cultures. Treatment of the cells with triclosan (0.5 ,g/ml) reduced both IFN-, production and MHC class II expression in human gingival fibroblast cultures. Similar inhibitory effects on IFN-, production and MHC class II expression were observed when the anti-inflammatory agent dexamethazone (1 ,M) was used. Conclusion: The present study further supports the view that the agent has an anti-inflammatory effect in addition to its antibacterial capacity. [source] Expression of keratinocyte growth factor and its receptor in odontogenic keratocystsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 5 2009Y. Suyama Background:, The mitotic activity of the epithelial cells of odontogenic keratocysts (OKCs) is greater than that of other odontogenic jaw cysts, and the mitotic activity of the epithelial cells decreases after marsupialization. Keratinocyte growth factor (KGF) interacts with its specific receptor (KGFR), and elicits the proliferation and/or differentiation of the various types of epithelial cells. The aim of this study was to investigate the expression of KGF/KGFR in OKCs before and after marsupialization. Methods:, The expression of KGF was immunohistochemically detected in the specimens of 16 OKCs and 11 dentigerous cysts before and after marsupialization. The expression of KGF mRNA was measured in the fibroblasts isolated from OKCs by real-time PCR. Results:, KGF was expressed in the epithelial cells and fibroblasts of 12 and seven of 16 OKC specimens, respectively. The intensity of the KGF expression in both the epithelial cells and the fibroblasts significantly decreased after marsupialization. KGFR was expressed throughout the epithelium in 15 of 16 OKC specimens, but the intensity of the KGFR expression did not change after marsupialization. The expression of KGF was detected in the epithelium of two of 11 dentigerous cyst specimens, but not in the fibroblasts before marsupialization. Real-time PCR revealed that recombinant human interleukin (IL)-1, increased the expression of KGF mRNA in the fibroblasts isolated from OKCs. Conclusion:, KGF/KGFR signaling may play a crucial role in the epithelial cells of OKCs. Furthermore, the expression of KGF in the fibroblasts of OKCs is regulated by IL-1,. [source] Decreased expression of ,2 integrin in fibroblasts isolated from cyclosporin A- induced gingival overgrowth in ratsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2003Masatoshi Kataoka Objectives:, Cyclosporin A (CsA), an immunosuppressive agent, induces fibrous gingival overgrowth through reduction of collagen phagocytosis by fibroblasts. Distinct receptors are involved in the binding of collagen to fibroblasts in collagen phagocytosis, and ,2,1 integrin serves as a specific receptor for type I collagen on fibroblasts. To elucidate the role of ,2,1 integrin in CsA-induced gingival overgrowth, we investigated collagen phagocytosis and ,2,1 integrin expression in rat gingival fibroblasts. Materials and methods:, Fibroblats were isolated from gingiva of rats fed a powdered diet containing or lacking CsA for 30 d. Flow cytometric analysis were performed to measure the collagen phagocytosis and the ,2 integrin expression in fibroblasts. Furthermore, total RNAs were isolated from fibroblasts, and the reverse transcriptase-polymerase chain reaction was employed to investigate the mRNA levels of ,2 integrin. Results:,In vitro collagen phagocytosis assay revealed that CsA-treated and control fibroblasts contained a mean of 13.5% and 36.1% phagocytic cells, respectively. CsA-treated fibroblasts had 28% lower expression of ,2 integrin than that of control. and mRNA expression of ,2 integrin in CsA-treated fibroblasts was apparently lower than in the controls, but the mRNA expression of ,1 integrin was not affected. Conclusion:, These findings suggest that one etiological factor of gingival overgrowth may be inhibition of collagen phagocytosis by reducing ,2 integrin expression in gingival fibroblasts. [source] Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor systemMOLECULAR ORAL MICROBIOLOGY, Issue 1 2003J. Hatakeyama We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin-8 (IL-8) responses of the cells to Salmonella lipopolysaccharide, a water-soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll-like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR-related molecules, i.e. TLR2, TLR4, MD-2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL-8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL-8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2- and TLR4-independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti-CD14 MAb. [source] Loss of ,1 integrin in mouse fibroblasts results in resistance to skin scleroderma in a mouse modelARTHRITIS & RHEUMATISM, Issue 9 2009Shangxi Liu Objective Activated adhesive signaling is a hallmark of fibroblasts isolated from the scars of scleroderma (systemic sclerosis) lesions. Beta-1 integrin plays a key role in adhesive signaling. The aim of the present study was to examine the role of ,1 integrin in a mouse model of skin scleroderma using mice bearing a fibroblast-specific deletion of ,1 integrin. Methods Cutaneous sclerosis was induced by subcutaneous injection of bleomycin. Control groups were treated with phosphate buffered saline. Mice bearing a fibroblast-specific deletion of ,1 integrin and control mice were investigated. Dermal thickness, collagen production, and the number of ,-smooth muscle actin,positive cells were determined. The quantity of the collagen-specific amino acid hydroxyproline was also measured. Results Bleomycin treatment induced marked cutaneous thickening and fibrosis in control mice. Conversely, the deletion of ,1 integrin resulted in resistance to bleomycin-induced fibrosis. Conclusion Expression of ,1 integrin by fibroblasts is required for fibrogenesis. Inhibition of ,1 integrin may be a viable method to alleviate the development of cutaneous sclerosis. [source] Expression and regulation of connective tissue growth factor by transforming growth factor , and tumour necrosis factor , in fibroblasts isolated from strictures in patients with Crohn's diseaseBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 10 2006D. Beddy Background: Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and extracellular matrix production. Fibroblasts may initiate stricture formation in Crohn's disease through overexpression of CTGF. Stricturing that occurs in patients with Crohn's disease after treatment with anti-tumour necrosis factor (TNF) , may be due to dysregulation of CTGF homeostasis. The aim of this study was to examine CTGF expression and regulation in fibroblasts isolated from patients with Crohn's disease. Methods: Fibroblasts were isolated by a primary explant technique from serosal biopsies of strictured segments of bowel in eight patients undergoing resection for Crohn's disease and from normal colon in seven patients having resection for benign or malignant colorectal disease. Cells were stimulated with transforming growth factor (TGF) , and TNF-,. CTGF protein and mRNA expression were measured by western blotting and real-time polymerase chain reaction respectively. Results: Mean(s.d.) CTGF protein expression in strictured Crohn's fibroblasts was higher than that in normal fibroblasts (56·5(9·7) versus 17·0(10·0) respectively; P = 0·011). In normal and strictured Crohn's fibroblasts, culture with TGF-, increased CTGF protein and mRNA expression. Co-culture of normal fibroblasts with TNF-, suppressed TGF-,-stimulated CTGF expression. Conclusion: Increased expression of CTGF in strictured Crohn's fibroblasts underlies its role in fibrosis. TNF-, suppresses fibrosis by downregulating fibroblast CTGF expression, an effect that may be lost following anti-TNF-, treatment, thereby promoting stricture formation. Copyright © 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] | ||||||||||