FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2134007677> ?p ?o ?g. }

• W2134007677 endingPage "6545" @default.
• W2134007677 startingPage "6530" @default.
• W2134007677 abstract "This study aims to understand the role of the matrix polysaccharide hyaluronan (HA) in influencing fibroblast proliferation and thereby affecting wound healing outcomes. To determine mechanisms that underlie scarred versus scar-free healing, patient-matched dermal and oral mucosal fibroblasts were used as models of scarring and non-scarring fibroblast phenotypes. Specifically, differences in HA generation between these distinct fibroblast populations have been examined and related to differences in transforming growth factor-β1 (TGF-β1)-dependent proliferative responses and Smad signaling. There was a differential growth response to TGF-β1, with it inducing proliferation in dermal fibroblasts but an anti-proliferative response in oral fibroblasts. Both responses were Smad3-dependent. Furthermore, the two fibroblast populations also demonstrated differences in their HA regulation, with dermal fibroblasts generating increased levels of HA, compared with oral fibroblasts. Inhibition of HA synthesis in dermal fibroblasts was shown to abrogate the TGF-β1-mediated induction of proliferation. Inhibition of HA synthesis also led to an attenuation of Smad3 signaling in dermal fibroblasts. Microarray analysis demonstrated no difference in the genes involved in TGF-β1 signaling between dermal and oral fibroblasts, whereas there was a distinct difference in the pattern of genes involved in HA regulation. In conclusion, these two distinct fibroblast populations demonstrate a differential proliferative response to TGF-β1, which is associated with differences in HA generation. TGF-β1 regulates proliferation through Smad3 signaling in both fibroblast populations; however, it is the levels of HA generated by the cells that influence the outcome of this response. This study aims to understand the role of the matrix polysaccharide hyaluronan (HA) in influencing fibroblast proliferation and thereby affecting wound healing outcomes. To determine mechanisms that underlie scarred versus scar-free healing, patient-matched dermal and oral mucosal fibroblasts were used as models of scarring and non-scarring fibroblast phenotypes. Specifically, differences in HA generation between these distinct fibroblast populations have been examined and related to differences in transforming growth factor-β1 (TGF-β1)-dependent proliferative responses and Smad signaling. There was a differential growth response to TGF-β1, with it inducing proliferation in dermal fibroblasts but an anti-proliferative response in oral fibroblasts. Both responses were Smad3-dependent. Furthermore, the two fibroblast populations also demonstrated differences in their HA regulation, with dermal fibroblasts generating increased levels of HA, compared with oral fibroblasts. Inhibition of HA synthesis in dermal fibroblasts was shown to abrogate the TGF-β1-mediated induction of proliferation. Inhibition of HA synthesis also led to an attenuation of Smad3 signaling in dermal fibroblasts. Microarray analysis demonstrated no difference in the genes involved in TGF-β1 signaling between dermal and oral fibroblasts, whereas there was a distinct difference in the pattern of genes involved in HA regulation. In conclusion, these two distinct fibroblast populations demonstrate a differential proliferative response to TGF-β1, which is associated with differences in HA generation. TGF-β1 regulates proliferation through Smad3 signaling in both fibroblast populations; however, it is the levels of HA generated by the cells that influence the outcome of this response. Scarring is an important response to tissue injury that facilitates repair through replacement of damaged tissue with fibrous tissue. In the context of dermal injury, this restores tissue strength and aids in the repair of structural defects. However, in some injuries, deposition of scar tissue may have adverse consequences. Following injury to internal organs, repair by scarring can initiate a cascade of events that continue unabated leading to progressive accumulation of fibrous tissue and eventually resulting in loss of organ function (1Eckes B. Zigrino P. Kessler D. Holtkotter O. Shephard P. Mauch C. Krieg T. Matrix Biol. 2000; 19: 325-332Crossref PubMed Scopus (197) Google Scholar, 2Gabbiani G. J. Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1223) Google Scholar). End-stage organ dysfunction because of progressive fibrosis comprises a wide range of disorders, including congestive cardiac failure, chronic kidney disease, pulmonary fibrosis, and liver cirrhosis, well known causes of worldwide death and disability (3Green F.H. Chest. 2002; 122: 334-339Abstract Full Text Full Text PDF Scopus (82) Google Scholar, 4Chapman H.A. J. Clin. Investig. 2004; 113: 148-157Crossref PubMed Scopus (0) Google Scholar, 5Eddy A.A. Pediatr. Nephrol. 2000; 15: 290-301Crossref PubMed Scopus (540) Google Scholar, 6Bedossa P. Paradis V. J. Pathol. 2003; 200: 504-515Crossref PubMed Scopus (204) Google Scholar, 7Anversa P. Li P. Zhang X. Olivetti G. Capasso J.M. Cardiovasc. Res. 1993; 27: 145-157Crossref PubMed Scopus (114) Google Scholar, 8Francis G.S. McDonald K. Chu C. Cohn J.N. Am. J. Cardiol. 1995; 75: A11-A16Abstract Full Text PDF PubMed Scopus (46) Google Scholar). Fibroblasts are the most abundant cell type in connective tissue, and they play a central role in extracellular matrix (ECM) 3The abbreviations used are:ECMextracellular matrixTGF-β1transforming growth factor-β1FITCfluorescein isothiocyanateHAhyaluronanPIpropidium iodideGAPDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionQPCRquantitative PCRFBSfetal bovine serumPBSphosphate-buffered salineHAPBHA-binding proteinELISAenzyme-linked immunosorbent assay4MU4-methylumbelliferoneFACSfluorescence-activated cell sortersiRNAshort interfering RNATEtrypsin extractCEcell extractTBSTris-buffered salineBisTris2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diolIQRinter-quartile rangeMAPKmitogen-activated protein kinase.3The abbreviations used are:ECMextracellular matrixTGF-β1transforming growth factor-β1FITCfluorescein isothiocyanateHAhyaluronanPIpropidium iodideGAPDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionQPCRquantitative PCRFBSfetal bovine serumPBSphosphate-buffered salineHAPBHA-binding proteinELISAenzyme-linked immunosorbent assay4MU4-methylumbelliferoneFACSfluorescence-activated cell sortersiRNAshort interfering RNATEtrypsin extractCEcell extractTBSTris-buffered salineBisTris2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diolIQRinter-quartile rangeMAPKmitogen-activated protein kinase. remodeling and wound contraction during tissue repair. In response to tissue injury, they proliferate, migrate to the site of injury, and differentiate into their active form, myofibroblasts (2Gabbiani G. J. Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1223) Google Scholar, 9Grinnell F. J. Cell Biol. 1994; 124: 401-404Crossref PubMed Scopus (968) Google Scholar, 10Krieg T. Heckmann M. Recent Prog. Med. 1989; 80: 594-598PubMed Google Scholar). These myofibroblasts are then involved in wound contraction and ECM synthesis and turnover (11Tomasek J.J. Gabbiani G. Hinz B. Chaponnier C. Brown R.A. Nat. Rev. 2002; 3: 349-363Crossref Scopus (3068) Google Scholar). They are also considered to be the key effector cells in fibrotic disease, and increased activity and proliferation of resident fibroblasts are central to fibrosis in all tissues (2Gabbiani G. J. Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1223) Google Scholar). The cytokine transforming growth factor-β1 (TGF-β1) is a known mediator of fibroblast-myofibroblast differentiation, and it mainly elicits its effects through the Smad signal transduction pathway (12Vaughan M.B. Howard E.W. Tomasek J.J. Exp. Cell Res. 2000; 257: 180-189Crossref PubMed Scopus (397) Google Scholar). It also influences a range of other cellular processes, including migration and proliferation, and its release initiates a sequence of events that are crucial in tissue repair, including chemoattraction of inflammatory cells, induction of angiogenesis, and regulation of inflammatory mediators (13Ten Dijke P. Goumans M.J. Itoh F. Itoh S. J. Cell. Physiol. 2002; 191: 1-16Crossref PubMed Scopus (376) Google Scholar, 14Moustakas A. Pardali K. Gaal A. Heldin C.H. Immunol. Lett. 2002; 82: 85-91Crossref PubMed Scopus (420) Google Scholar, 15Letterio J.J. Roberts A.B. Annu. Rev. Immunol. 1998; 16: 137-161Crossref PubMed Scopus (1655) Google Scholar, 16Border W.A. Noble N.A. N. Engl. J. Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2994) Google Scholar, 17Diegelmann R.F. Evans M.C. Front. Biosci. 2004; 9: 283-289Crossref PubMed Scopus (1446) Google Scholar). However, aberrant expression of TGF-β1 has been demonstrated in virtually every type of fibrotic disease, and several reports have established that increased TGF-β1 expression directly correlates with progressive tissue fibrosis and disease progression (16Border W.A. Noble N.A. N. Engl. J. Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2994) Google Scholar, 18Border W.A. Noble N.A. J. Clin. Investig. 1995; 96: 655-656Crossref PubMed Scopus (48) Google Scholar, 19Shihab F.S. Yamamoto T. Nast C.C. Cohen A.H. Noble N.A. Gold L.I. Border W.A. J. Am. Soc. Nephrol. 1995; 6: 286-294PubMed Google Scholar, 20Border W.A. Ruoslahti E. J. Clin. Investig. 1992; 90: 1-7Crossref PubMed Scopus (1042) Google Scholar). In this context, a better understanding of factors involved in TGF-β1 regulation and its influence on fibroblast behavior is crucial in determining the pathogenic mechanisms underlying progressive fibrosis and may provide novel insights into developing new therapeutic strategies.In comparison with other tissues, wounds in the oral mucosa are clinically distinguished in that they heal without notable scar formation resulting in complete regeneration of tissue structure and restoration of function. Several lines of evidence indicate that although the oral mucosal environment (in comparison to other environments) demonstrates differences in inflammatory cell infiltrate and cytokine gene expression, tissue from the oral mucosa also possesses intrinsic differences that can account for its differential wound healing profile (21Meran S. Steadman R. Phillips A.O. J. Am. Soc. Nephrol. 2006; 17: 126Google Scholar, 22Meran S. Stephens P. Thomas D.W. Phillips A.O. Steadman R. J. Biol. Chem. 2007; 282: 25687-25697Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 23Szpaderska A.M. Zuckerman J.D. DiPietro L.A. J. Dent. Res. 2003; 82: 621-626Crossref PubMed Scopus (271) Google Scholar, 24Stephens P. Davies K.J. al-Khateeb T. Shepherd J.P. Thomas D.W. J. Dent. Res. 1996; 75: 1358-1364Crossref PubMed Scopus (90) Google Scholar, 25Stephens P. Davies K.J. Occleston N. Pleass R.D. Kon C. Daniels J. Khaw P.T. Thomas D.W. Br. J. Dermatol. 2001; 144: 229-237Crossref PubMed Scopus (112) Google Scholar). In an attempt to delineate the mechanisms that differentiate between scarring versus scar-free repair, we have used patient-matched oral mucosal and dermal fibroblasts, as models of non-scarring and scarring fibroblast phenotypes, respectively, to attempt to identify the mechanisms that could be exploited to prevent scarring following tissue injury.We have previously demonstrated clear differences between oral mucosal and dermal fibroblasts that can account for the differential wound healing profiles of the two tissues. Fibroblasts derived from the oral mucosa have been shown to demonstrate increased migration, experimental wound repopulation, and extracellular matrix re-organization as compared with fibroblasts derived from the dermis of the same individual (24Stephens P. Davies K.J. al-Khateeb T. Shepherd J.P. Thomas D.W. J. Dent. Res. 1996; 75: 1358-1364Crossref PubMed Scopus (90) Google Scholar, 25Stephens P. Davies K.J. Occleston N. Pleass R.D. Kon C. Daniels J. Khaw P.T. Thomas D.W. Br. J. Dermatol. 2001; 144: 229-237Crossref PubMed Scopus (112) Google Scholar). We have also shown recently that these same fibroblast populations demonstrate intrinsic differences in their ability to differentiate in response to the pro-fibrotic cytokine TGF-β1. Although dermal fibroblasts readily differentiate in response to TGF-β1, oral mucosal fibroblasts are resistant to TGF-β1-driven myofibroblastic differentiation. Furthermore, we demonstrated that the matrix polysaccharide hyaluronan (HA) plays a pivotal role in regulating TGF-β1-driven cellular differentiation in that it facilitates fibroblast-myofibroblast transition (21Meran S. Steadman R. Phillips A.O. J. Am. Soc. Nephrol. 2006; 17: 126Google Scholar, 22Meran S. Stephens P. Thomas D.W. Phillips A.O. Steadman R. J. Biol. Chem. 2007; 282: 25687-25697Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The purpose of this study was to expand these observations by comparing differences in other TGF-β1-mediated cell responses in scarring and non-scarring fibroblast phenotypes. Furthermore, this work aimed to test the general applicability of the hypothesis that intrinsic differences in HA generation in the two fibroblast populations is the key to determining cellular responses and influencing wound healing outcomes.The data presented here show that fibroblasts derived from the dermis and patient-matched oral mucosa demonstrate a differential proliferative response to TGF-β1. Although TGF-β1 stimulates proliferation in dermal fibroblasts, it inhibits DNA synthesis and induces an anti-proliferative response in oral mucosal fibroblasts. The two fibroblast populations also demonstrate differences in their HA regulation, with dermal fibroblasts generating increased levels of HA as compared with oral fibroblasts. Furthermore, the data demonstrate that TGF-β1 regulates proliferation through Smad3 signaling in both fibroblast populations. However, the proliferative response to TGF-β1 in dermal fibroblasts appears to be directly linked to the increased levels of HA generated by these cells.EXPERIMENTAL PROCEDURESMaterials—All reagents were from Sigma unless otherwise stated. Reverse transcription and QPCR reagents and primers were purchased from Invitrogen and Applied Biosystems (Cheshire, UK). Radioisotopes were purchased from Amersham Biosciences.Cell Culture—Four donor-matched samples of dermal and oral mucosal fibroblasts were obtained by biopsy from consenting adults undergoing routine minor surgery, and ethical approval for the biopsies was obtained from the South East Wales Research Ethics Committee. The cells were isolated as described previously (25Stephens P. Davies K.J. Occleston N. Pleass R.D. Kon C. Daniels J. Khaw P.T. Thomas D.W. Br. J. Dermatol. 2001; 144: 229-237Crossref PubMed Scopus (112) Google Scholar) and cultured in Dulbecco's modified Eagle's medium and F-12 medium containing 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin supplemented with 10% fetal bovine serum (FBS) (Biologic Industries Ltd., Cumbernauld, UK). The cells were maintained at 37 °C in a humidified incubator in an atmosphere of 5% CO2, and fresh growth medium was added to the cells every 3-4 days until confluent. The cells were incubated in serum-free medium for 48 h before use in experiments, and all experiments were done under serum-free conditions unless otherwise stated. All experiments were undertaken using cells at passage 6-10 and performed on confluent cultures except for those examining proliferation. These experiments used subconfluent cells to allow for cell growth.Analysis of Cell Proliferation[3H]Thymidine Proliferation Assay—Fibroblast proliferation was first assessed by the incorporation of d-[3H]thymidine into DNA. Cells were grown in 35-mm dishes and assessed at subconfluence. Metabolic labeling was performed by incubation with 1 μCi/ml d-[3H]thymidine for 24 h. The medium was then discarded, and the cells were washed repeatedly with PBS containing 1 mm thymidine prior to fixing with 500 μl of 5% trichloroacetic acid containing 1 mm thymidine at 4 °C for 1 h. The cell layer was extracted by incubation with 1 ml of 0.1 m NaOH at 20 °C for 24 h and neutralized with 0.1 m HCl. The radioactivity was determined by β-counting on a Packard Tri-Carb 1900 liquid scintillation analyzer, and the results are represented as disintegrations/min.alamarBlue™ Proliferation Assay—Analysis of cell growth was also assessed by the commercial alamarBlue™ assay (BIO-SOURCE), which is designed to measure cell viability and number. The assay utilizes an oxidation-reduction indicator that fluoresces in response to chemical reduction of growth medium resulting from cell growth and metabolism and demonstrates a linear relationship between the magnitude of fluorescence and cell number and viability. For this assay cells were grown in 35-mm dishes and assessed at subconfluence following a 48-h period of growth arrest. The cells were then stimulated with 10 ng/ml TGF-β1. Following 24 h, 10% alamarBlue was added to the medium for 1 h at 37 °C. 100-μl aliquots of the conditioned medium were removed and added to a clear 96-well plate. Subsequently fluorescence was measured in a Fluostar optima fluorescence meter (BMG Lab Technologies) with excitation wavelength at 540 nm and emission wavelength at 590 nm; and the results are expressed as arbitrary fluorescence units.Analysis of Cell ApoptosisFluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide were used to confirm the presence of apoptotic cells in in vitro cultures. Cells were grown to confluence in 35-mm dishes, washed in PBS, and then harvested using trypsin/EDTA. The cells were then resuspended in 100 μl of the reaction mix made up of 90 μl of distilled water, 10 μl of 10× binding buffer (100 mm HEPES, 1.5 m NaCl, 50 mm KCl, 10 mm MgCl2, and 18 mm CaCl2, pH 7.4), 5 μl of FITC-annexin-V (Pharmingen), and 5 μl of propidium iodide (PI) (Pharmingen). The cells were incubated in the dark for 20 min at room temperature, centrifuged, washed in PBS, and resuspended in 1× binding buffer. The samples were analyzed immediately by flow cytometry (FACSCalibur). The viable cells were annexin-V/PI-negative, and cells in early apoptosis were annexin-V-positive and PI-negative, and cells in late apoptosis/necrosis were annexin-V/PI-positive.Cell Cycle AnalysisThe distribution of cells within the cell cycle was determined by propidium iodide incorporation into DNA. Cells were grown in 35-mm dishes and assessed at subconfluence. Briefly, cells were harvested by treatment with trypsin/EDTA, pelleted by centrifugation, and then washed in PBS. The cells were then resuspended in cold 70% ethanol and maintained at 4 °C for 16 h. Subsequently, the cells were centrifuged, washed in PBS, and then resuspended in 300 μl of binding buffer and 10 μl of 50 μg/ml PI solution along with 5 μl/sample RNase (10 mg/ml stock). The samples were then incubated at 37 °C for 30 min prior to FACS analysis. The percentage of cells in the different phases of the cell cycle were quantified using Cylchred version 1.0.2 software (Terry Hoy, University Hospital of Wales, Cardiff, UK).Immunoblotting/Western AnalysisWestern blot analysis was used to assess expression of phosphorylated Smad2 and Smad3. Cells were grown to confluence in 35-mm dishes and rinsed with cold PBS. Cells were then lysed using 1% protease inhibitor mixture, 1% phenylmethylsulfonyl fluoride, and 1% sodium orthovanadate in RIPA lysis buffer (Santa Cruz Biotechnology). The samples were scraped, collected, and centrifuged at 2500 rpm for 10 min. The supernatant was collected, and protein concentrations were determined by Bradford assay, and the samples were stored at -70 °C until use. Equal amounts of protein were mixed with equal volumes of reducing SDS sample buffer and denatured for 5 min at 95 °C before loading onto 10% SDS-polyacrylamide gels. Electrophoresis was carried out under reducing conditions at 150 V for 1 h, and the separated proteins were then transferred at 150 V over 90 min to a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with Tris-buffered saline (TBS) containing 5% nonfat powdered milk for 1 h and then incubated with the primary antibody (anti-phosphorylated Smad3 or anti-phosphorylated Smad2, 1:500 dilution in TBS, host:rabbit) at 4 °C overnight (Cell Signaling Technology, Danvers, MA). Expression of GAPDH was analyzed as a control to ensure equal loading (anti-GAPDH, 1:1000 dilution in TBS, host:rabbit). The blots were subsequently washed with TBS containing 1% Tween and then incubated with the secondary antibody for 1 h at room temperature (anti-rabbit IgG:horseradish peroxidase, 1:12,000 dilution in TBS). Proteins were visualized using enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions. The intensity of the bands was quantified by densitometry and corrected for the expression of GAPDH.Analysis of 3H-Radiolabeled HACells were grown to full confluence in T75 flasks. Metabolic labeling was performed by incubation with 20 μCi/ml d-[3H]glucosamine hydrochloride for the indicated times. The culture medium was then removed, and the cells were washed with PBS. The wash and medium were combined to form the conditioned medium extract (CM). The CM was then treated with an equal volume of 200 μg/ml Pronase in 100 mm Tris-HCl, pH 8.0, 0.05% sodium azide for 24 h. To remove any CD44-associated HA, the cells were treated with 10 μg/ml trypsin in PBS for 10 min at room temperature; this was designated the trypsin extract (TE). The TE was then treated with an equal volume of 200 μg/ml Pronase for 24 h. The cells remaining in the culture flask were incubated directly with 100 μg/ml Pronase for 24 h. The supernatant was decanted and designated the cell extract (CE).Each extract was passed over DEAE-Sephacel ion exchange columns equilibrated with 8 m urea in 20 mm BisTris buffer, pH 6, containing 0.2% Triton X-100. This removed any low molecular weight peptides and unincorporated radiolabel. HA was eluted in 8 m urea buffer containing 0.3 m NaCl. Each sample was split into two, and the HA was precipitated with 3 volumes of 1% potassium acetate in 95% ethanol in the presence of 50 μg/ml of each HA, heparin, and chondroitin sulfate as co-precipitants. The first half of each sample was resuspended in 500 μl of 4 m guanidine buffer and analyzed on a Sephacryl S-500 column equilibrated with 4 m guanidine buffer. To confirm that the chromatography profile generated was the result of radiolabeled HA, the second half of each sample was digested at 37 °C overnight with 1 unit of Streptomyces hyalurolyticus hyaluronidase (ICN Pharmaceuticals Ltd.) in 200 μl of 20 mm sodium acetate, pH 6.0, containing 0.05% sodium azide and 0.15 m sodium chloride. The sample was then mixed with an equal volume of 4 m guanidine buffer and analyzed on the same Sephacryl S-500 column equilibrated with 4 m guanidine buffer. To produce the chromatography profile the 3H activity for each half of the sample was normalized and corrected for dilution, and then the hyaluronidase-resistant counts were subtracted. The chromatography profiles depict only the hyaluronidase-sensitive activity in each fraction plotted against fraction number. The column was calibrated with [3H]glucosamine hydrochloride, Mr 215; [35S]chondroitin sulfate glycosaminoglycans, Mr 25 × 103; decorin, Mr 10 × 104; and [35S]versican, Mr 1.3 × 106. The radioactivity was determined by β-counting on a Packard Tri-Carb 1900 liquid scintillation analyzer, and the results are represented as disintegrations/min.Determination of HA ConcentrationCells were grown to confluence in 35-mm dishes, and the HA concentration in the cell culture supernatant was determined using a commercially available enzyme-linked HA-binding protein assay (HA “Chugai” quantitative test kit; Congenix, Petersborough, UK). The assay used microwells coated with a highly specific HA-binding protein (HABP) from bovine cartilage to capture HA and an enzyme-conjugated version of HABP to detect and measure HA in the samples. Briefly, diluted samples and HA reference solutions were incubated in HABP-coated microwells allowing binding of the HA in the samples to the immobilized HABP. The wells were then washed, and HABP conjugated with horseradish peroxidase was added to the wells forming complexes with bound HA. Following a second washing step, a chromogenic substrate (TMB:H2O2) was added to develop a colored reaction. Stopping solution was added to the wells, and the intensity of the resulting color was measured in optical density units using a spectrophotometer at 450 nm. HA concentrations were calculated by comparing the absorbance of the sample against a reference curve prepared from the reagent blank and five HA reference solutions (50, 100, 200, 500, and 800 ng/ml) included in the kit. The assay is sensitive to 10 ng/ml, with no cross-reactivity with other glycosaminoglycan compounds.Determination of TGF-β1 ProductionTGF-β1 concentration in the culture supernatant was determined by specific enzyme-linked immunosorbent assay (ELISA) (R & D Systems Europe Ltd.). This assay has less than 1% cross-reactivity with TGF-β2 and TGF-β3. Briefly, high protein-binding 96-well plates were coated with TGF-β capture antibody (2 μg/ml in PBS) overnight at room temperature. Wells were washed, and the plate was incubated for 1 h at room temperature with block buffer (5% Tween 20, 5% sucrose in PBS) and then washed before addition of TGF-β standards and cell culture supernatant samples. Cells were grown to confluence in 35-mm dishes and growth-arrested in serum-free medium for 48 h. Subsequently the medium was changed to medium containing 0.5 mm 4MU or serum-free medium alone, and the incubations were continued for a further 24 h. 20 μl of 1 m HCl was then added to 100-μl samples of cell supernatants to acid-activate latent TGF-β1; and the samples were incubated for 10 min at room temperature. The acid-activated samples were neutralized with 20 μl of 1.2 m NaOH, 0.5 m HEPES. In parallel experiments, an equal number of 100-μl cell supernatant samples were analyzed without acid activation to quantify active rather than total TGF-β1 concentrations. Samples and standards were then added to the plate and incubated for 2 h at room temperature. The plate was washed, and detection antibody was added at 300 ng/ml in reagent diluent (1.4% delipidized bovine serum albumin/Tween 20 in PBS). The plate was then incubated for a further 2 h at room temperature and washed again. Streptavidin-horseradish peroxidase (0.5% in reagent diluent) was added to each well, the plate was incubated in the dark for 20 min and then washed. Substrate solution (TMB/H2O2) was added to the wells, and the plate was incubated in the dark for 20 min before the addition of stop solution. The intensity of the resulting color was measured in optical density units using a spectrophotometer at 450 nm. TGF-β1 concentration was calculated by comparing the absorbance of the sample against a reference curve prepared from the reagent blank and the standards.Cytotoxicity AssayCytotoxicity of 4MU was assessed by the alamarBlue™ assay. Subconfluent dermal fibroblasts were incubated for 24 h in serum-free medium alone or serum-free medium containing 1, 0.5, 0.4, 0.3, or 0.2 mm 4MU. Following 24 h 10% alamarBlue was added to the medium for 1 h at 37 °C. 100-μl aliquots of the conditioned medium were removed and added to a clear 96-well plate. Subsequently fluorescence was measured in a Fluostar optima fluorescence meter (BMG Lab Technologies) with excitation wavelength at 540 nm and emission wavelength at 590 nm; and the results are expressed as arbitrary fluorescence units.Smad3 siRNA TransfectionTransient transfection of dermal and oral fibroblasts with specific siRNA nucleotides targeting Smad3 expression was performed using siPORT amine transfection reagent (Ambion Ltd., Huntington, UK) in accordance with the manufacturer's protocol. Briefly, 8 μl of transfection agent was diluted in 100 μl of Opti-MEM reduced growth medium (Invitrogen) and left to incubate at room temperature for 10 min. Meanwhile, the specific Smad3 siRNA oligonucleotides were diluted in Opti-MEM reduced growth medium to give a final concentration of 20 μm in a total volume of 100 μl. The transfection agent mix and siRNA mix were then combined and incubated at room temperature for a further 10 min. The newly formed transfection complexes (200 μl) were dispensed into empty wells of 6-well culture plates. To each well, 2.3 × 105 cells were then added so that the total volume in each well was 2500 μl. As a control, cells were transfected with negative control siRNA (a scrambled sequence that bears no homology to the human genome). The cells were then incubated at 37 °C with 5% CO2 for 24 h in medium supplemented with 10% FBS followed by a 24-h incubation in serum-free medium prior to experimentation.Reverse Transcription (RT) and Quantitative PCR (QPCR)RT-QPCR was used to assess Smad3, HAS2, versican, and aggrecan mRNA expression in oral mucosal and dermal fibroblasts. The cells were grown to confluence in 35-mm dishes and washed with PBS prior to lysis with tri-reagent and RNA purification according to the manufacturer's protocol. RT was performed using the random hexamer method. 1 μg of RNA was added to 1 μl of 100 μm random hexamers, 2 μl of 10× PCR buffer, and 2 μl of 0.1 m dithiothreitol. The solution was heated to 95 °C for 5 min followed by 4 °C for 2 min. 1 μl of (40 units/μl) ribonuclease inhibitor RNasin (Promega) and 1 μl of (200 units/μl) Superscript were added to each sample and mixed. The solution was incubated at 20 °C for 10 min, 42 °C for 40 min, and then 95 °C for 5 min on a GeneAmp PCR System 9700. As a negative control RT was performed with sterile H2O replacing the RNA sample.QPCR was performed using the 7900HT fast real time PCR system from Applied Bioscie" @default.
• W2134007677 created "2016-06-24" @default.
• W2134007677 creator A5010362194 @default.
• W2134007677 creator A5011609796 @default.
• W2134007677 creator A5011921917 @default.
• W2134007677 creator A5029931631 @default.
• W2134007677 creator A5042764711 @default.
• W2134007677 creator A5043026999 @default.
• W2134007677 creator A5088059961 @default.
• W2134007677 date "2008-03-01" @default.
• W2134007677 modified "2023-10-17" @default.
• W2134007677 title "Hyaluronan Facilitates Transforming Growth Factor-β1-mediated Fibroblast Proliferation" @default.
• W2134007677 cites W1605041458 @default.
• W2134007677 cites W1900562057 @default.
• W2134007677 cites W1964195356 @default.
• W2134007677 cites W1968952749 @default.
• W2134007677 cites W1982917185 @default.
• W2134007677 cites W1984760520 @default.
• W2134007677 cites W1988349464 @default.
• W2134007677 cites W1991301758 @default.
• W2134007677 cites W1992511421 @default.
• W2134007677 cites W1992635362 @default.
• W2134007677 cites W1994738588 @default.
• W2134007677 cites W1995806216 @default.
• W2134007677 cites W1997010716 @default.
• W2134007677 cites W1997237071 @default.
• W2134007677 cites W1998714482 @default.
• W2134007677 cites W2002867354 @default.
• W2134007677 cites W2009629707 @default.
• W2134007677 cites W2010463124 @default.
• W2134007677 cites W2014484446 @default.
• W2134007677 cites W2016193552 @default.
• W2134007677 cites W2030605936 @default.
• W2134007677 cites W2035391762 @default.
• W2134007677 cites W2037038224 @default.
• W2134007677 cites W2041150111 @default.
• W2134007677 cites W2042778072 @default.
• W2134007677 cites W2043898015 @default.
• W2134007677 cites W2045141910 @default.
• W2134007677 cites W2045580622 @default.
• W2134007677 cites W2051029508 @default.
• W2134007677 cites W2051696368 @default.
• W2134007677 cites W2056199187 @default.
• W2134007677 cites W2056501914 @default.
• W2134007677 cites W2058314672 @default.
• W2134007677 cites W2066291735 @default.
• W2134007677 cites W2069305370 @default.
• W2134007677 cites W2070454531 @default.
• W2134007677 cites W2071589283 @default.
• W2134007677 cites W2075075502 @default.
• W2134007677 cites W2076706080 @default.
• W2134007677 cites W2077388392 @default.
• W2134007677 cites W2078841581 @default.
• W2134007677 cites W2084092470 @default.
• W2134007677 cites W2085600594 @default.
• W2134007677 cites W2085889262 @default.
• W2134007677 cites W208674030 @default.
• W2134007677 cites W2090463334 @default.
• W2134007677 cites W2093024719 @default.
• W2134007677 cites W2095741501 @default.
• W2134007677 cites W2095867900 @default.
• W2134007677 cites W2096852597 @default.
• W2134007677 cites W2109935448 @default.
• W2134007677 cites W2114224461 @default.
• W2134007677 cites W2118121278 @default.
• W2134007677 cites W2119139128 @default.
• W2134007677 cites W2122081319 @default.
• W2134007677 cites W2125084288 @default.
• W2134007677 cites W2129379081 @default.
• W2134007677 cites W2131365724 @default.
• W2134007677 cites W2134213816 @default.
• W2134007677 cites W2139786796 @default.
• W2134007677 cites W2140524396 @default.
• W2134007677 cites W2142605112 @default.
• W2134007677 cites W2145261791 @default.
• W2134007677 cites W2151302132 @default.
• W2134007677 cites W2154645733 @default.
• W2134007677 cites W2156918892 @default.
• W2134007677 cites W2159657475 @default.
• W2134007677 cites W2164754162 @default.
• W2134007677 cites W2170296643 @default.
• W2134007677 cites W2170326305 @default.
• W2134007677 cites W2316067769 @default.
• W2134007677 cites W2335187016 @default.
• W2134007677 cites W2391160957 @default.
• W2134007677 cites W2409337244 @default.
• W2134007677 doi "https://doi.org/10.1074/jbc.m704819200" @default.
• W2134007677 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18174158" @default.
• W2134007677 hasPublicationYear "2008" @default.
• W2134007677 type Work @default.
• W2134007677 sameAs 2134007677 @default.
• W2134007677 citedByCount "115" @default.
• W2134007677 countsByYear W21340076772012 @default.
• W2134007677 countsByYear W21340076772013 @default.
• W2134007677 countsByYear W21340076772014 @default.
• W2134007677 countsByYear W21340076772015 @default.
• W2134007677 countsByYear W21340076772016 @default.
• W2134007677 countsByYear W21340076772017 @default.

• next