FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1541546534> ?p ?o ?g. }

• W1541546534 endingPage "2764" @default.
• W1541546534 startingPage "2753" @default.
• W1541546534 abstract "Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2−/− mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2−/− mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease. Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2−/− mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2−/− mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease. Idiopathic pulmonary fibrosis (IPF) constitutes the most devastating form of fibrotic idiopathic interstitial pneumonias.1Katzenstein AL Myers JL Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification.Am J Respir Crit Care Med. 1998; 157: 1301-1315Crossref PubMed Scopus (1177) Google Scholar, 2Demedts M Costabel U ATS/ERS international multidisciplinary consensus classification of the idiopathic interstitial pneumonias.Eur Respir J. 2002; 19: 794-796Crossref PubMed Scopus (132) Google Scholar It generally evolves toward a destructive and irreversible fibrosis, responsible for respiratory failure. The median survival is 2 to 5 years.3Swigris JJ Kuschner WG Kelsey JL Gould MK Idiopathic pulmonary fibrosis: challenges and opportunities for the clinician and investigator.Chest. 2005; 127: 275-283Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 4Raghu G Weycker D Edelsberg J Bradford WZ Oster G Incidence and prevalence of idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2006; 174: 810-816Crossref PubMed Scopus (992) Google Scholar Although substantial efforts led to considerable improvement in diagnosing the diseases and predicting the outcome, fibrotic lung diseases remain generally refractory to current available pharmacological therapies.4Raghu G Weycker D Edelsberg J Bradford WZ Oster G Incidence and prevalence of idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2006; 174: 810-816Crossref PubMed Scopus (992) Google Scholar, 5Scotton CJ Chambers RC Molecular targets in pulmonary fibrosis: the myofibroblast in focus.Chest. 2007; 132: 1311-1321Crossref PubMed Scopus (440) Google Scholar Interestingly, coagulation cascade activation is frequently observed in lungs of patients with fibrotic lung diseases. Enhanced expression of tissue factor (TF) and intra-alveolar fibrin deposition were demonstrated in lung biopsy specimens from patients with pulmonary fibrosis.6Hernandez-Rodriguez NA Cambrey AD Harrison NK Chambers RC Gray AJ Southcott AM duBois RM Black CM Scully MF McAnulty RJ Laurent GJ Role of thrombin in pulmonary fibrosis.Lancet. 1995; 346: 1071-1073Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 7Imokawa S Sato A Hayakawa H Kotani M Urano T Takada A Tissue factor expression and fibrin deposition in the lungs of patients with idiopathic pulmonary fibrosis and systemic sclerosis.Am J Respir Crit Care Med. 1997; 156: 631-636Crossref PubMed Scopus (119) Google Scholar, 8Gunther A Mosavi P Heinemann S Ruppert C Muth H Markart P Grimminger F Walmrath D Temmesfeld-Wollbruck B Seeger W Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome.Am J Respir Crit Care Med. 2000; 161: 454-462Crossref PubMed Scopus (295) Google Scholar, 9Idell S Coagulation, fibrinolysis, and fibrin deposition in acute lung injury.Crit Care Med. 2003; 31: S213-S220Crossref PubMed Google Scholar Procoagulant activity was demonstrated in bronchoalveolar lavage fluid (BALF) of patients with various forms of pulmonary fibrosis. Moreover, local procoagulant activity was demonstrated in patients with acute lung injury.6Hernandez-Rodriguez NA Cambrey AD Harrison NK Chambers RC Gray AJ Southcott AM duBois RM Black CM Scully MF McAnulty RJ Laurent GJ Role of thrombin in pulmonary fibrosis.Lancet. 1995; 346: 1071-1073Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 8Gunther A Mosavi P Heinemann S Ruppert C Muth H Markart P Grimminger F Walmrath D Temmesfeld-Wollbruck B Seeger W Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome.Am J Respir Crit Care Med. 2000; 161: 454-462Crossref PubMed Scopus (295) Google Scholar, 9Idell S Coagulation, fibrinolysis, and fibrin deposition in acute lung injury.Crit Care Med. 2003; 31: S213-S220Crossref PubMed Google Scholar, 10Gunther A Mosavi P Ruppert C Heinemann S Temmesfeld B Velcovsky HG Morr H Grimminger F Walmrath D Seeger W Enhanced tissue factor pathway activity and fibrin turnover in the alveolar compartment of patients with interstitial lung disease.Thromb Haemost. 2000; 83: 853-860PubMed Google Scholar The mechanistic link between coagulation activation and pulmonary fibrosis, however, remains elusive. Recent data suggest that protease-activated receptor 2 (PAR-2), a cellular receptor activated by (among others) coagulation factor (F)Xa, is an important player in fibrotic disease. PAR-2 triggers fibroproliferative responses in vitro11Wang J Zheng H Hollenberg MD Wijesuriya SJ Ou X Hauer-Jensen M Up-regulation and activation of proteinase-activated receptor 2 in early and delayed radiation injury in the rat intestine: influence of biological activators of proteinase-activated receptor 2.Radiat Res. 2003; 160: 524-535Crossref PubMed Scopus (38) Google Scholar, 12Masamune A Kikuta K Satoh M Suzuki N Shimosegawa T Protease-activated receptor-2-mediated proliferation and collagen production of rat pancreatic stellate cells.J Pharmacol Exp Ther. 2005; 312: 651-658Crossref PubMed Scopus (65) Google Scholar, 13Borensztajn K Stiekema J Nijmeijer S Reitsma PH Peppelenbosch MP Spek CA Factor Xa stimulates proinflammatory and profibrotic responses in fibroblasts via protease-activated receptor-2 activation.Am J Pathol. 2008; 172: 309-320Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar and is highly expressed in fibrotic disorders.12Masamune A Kikuta K Satoh M Suzuki N Shimosegawa T Protease-activated receptor-2-mediated proliferation and collagen production of rat pancreatic stellate cells.J Pharmacol Exp Ther. 2005; 312: 651-658Crossref PubMed Scopus (65) Google Scholar This study explores the role of blood coagulation factor driven PAR-2 activation in pulmonary fibrosis. We show that PAR-2 is up-regulated in the lung of IPF patients and that BALF of these patients displays increased procoagulant activity and enhances fibroblast survival. Using a mouse model of bleomycin-induced lung fibrosis, we show that PAR-2 deficiency is protective. Moreover, BALF of bleomycin-treated PAR-2 deficient mice less effectively induced fibroblast survival compared to BALF of wild-type treated mice; and bleomycin induced less coagulation activation in PAR-2 deficient mice. Taken together, PAR-2 seems to contribute to the progression of pulmonary fibrosis, and inhibition of the PAR-2-coagulation axis may be clinically relevant. Eight patients with biopsy-proven IPF (mean age 67 ± 6 yrs) were included in this study; IPF was defined according to international guidelines (American Thoracic Society/European Respiratory Society14American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias Joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001.Am J Respir Crit Care Med. 2002; 165: 277-304Crossref PubMed Scopus (3385) Google Scholar). Pathology specimens were judged according to the criteria described by Katzenstein and Myers.1Katzenstein AL Myers JL Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification.Am J Respir Crit Care Med. 1998; 157: 1301-1315Crossref PubMed Scopus (1177) Google Scholar In the IPF patients, bronchoalveolar lavage was performed as part of the routine work-up; the procedure of bronchoalveolar lavage was previously described.15Rijneveld AW Florquin S Bresser P Levi M De Waard V Lijnen R Van Der Zee JS Speelman P Carmeliet P Van Der Poll T Plasminogen activator inhibitor type-1 deficiency does not influence the outcome of murine pneumococcal pneumonia.Blood. 2003; 102: 934-939Crossref PubMed Scopus (74) Google Scholar BALF from nine healthy volunteers (mean age 32 ± 8 yrs) who had participated in a previously reported study15Rijneveld AW Florquin S Bresser P Levi M De Waard V Lijnen R Van Der Zee JS Speelman P Carmeliet P Van Der Poll T Plasminogen activator inhibitor type-1 deficiency does not influence the outcome of murine pneumococcal pneumonia.Blood. 2003; 102: 934-939Crossref PubMed Scopus (74) Google Scholar served as controls. From five patients, enough BALF was available to study in vitro stimulation of human lung fibroblast. Control BALF was obtained from healthy volunteers. The study was approved by the Institutional Scientific and Ethics committees. Written informed consent was obtained from all subjects. The levels of thrombin-antithrombin and that of soluble TF were measured in the BALF of IPF patients and controls using specific commercially available enzyme-linked immunosorbent assays (thrombin–antithrombin complex: Behringwerke AG, Marburg, Germany; soluble TF: American Diagnostics, Greenwich, CT) following the manufacturer's instructions. Ten-week-old wild-type C57Bl/6 mice were purchased from Charles River (Someren, the Netherlands). PAR-2 deficient (PAR-2−/−) C57Bl/6 mice were originally provided by Jackson Laboratories (Maine) and bred at the animal care facility of the Academic Medical Center. All mice were maintained according to institutional guidelines. Animal procedures were carried out in compliance with the Institutional Standards for Humane Care and Use of Laboratory Animals. The Animal Care and Use Committee of the Academic Medical Center (Amsterdam, the Netherlands) approved all experiments. In each experimental group, eight mice were used. For baseline characteristics, four mice per genotype were used. Bleomycin sulfate (1 mg/kg body weight in 45 μl of saline as described in16Howell DC Goldsack NR Marshall RP McAnulty RJ Starke R Purdy G Laurent GJ Chambers RC Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2001; 159: 1383-1395Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar; Sigma, St-Louis, MO) was administrated by a single intratracheal injection under anesthesia. Control animals received an intratracheal saline injection. Mice were sacrificed 7 and 14 days after bleomycin instillation. Bronchoalveolar lavage was performed by instilling four times 0.3 ml aliquots of saline by a 22-gauge Abbocath-T catheter into the trachea via a midline incision.17Leemans JC Vervoordeldonk MJ Florquin S van Kessel KP van der Poll T Differential role of interleukin-6 in lung inflammation induced by lipoteichoic acid and peptidoglycan from Staphylococcus aureus.Am J Respir Crit Care Med. 2002; 165: 1445-1450Crossref PubMed Scopus (89) Google Scholar Total BALF cell numbers were assessed with a Burker-Turk hemocytometer (Emergo, Landsmeer, The Netherlands). Briefly, cells were pelleted by centrifugation, then resuspended in PBS and counted according to the manufacturer's recommendations. Differential BALF cell counts were performed by cytospins prepared by centrifuging 100 μl of BALF suspensions containing 200,000 cells/ml. Slides were air dried and stained with Giemsa. Cells were identified as macrophages/monocytes, neutrophils, or lymphocytes, and data were expressed in absolute numbers. The right lung of each animal was excised 7 or 14 days after bleomycin or saline treatment. Following fixation, entire mouse lungs were embedded in paraffin. Four-micron thick sections were stained with H&E, Masson's trichrome, and Sirius red, according to routine procedures. In H&E-stained lung sections, three systems were used to assess fibrosis (see Table 118Ashcroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1084) Google Scholar, 19Theiss AL Fuller CR Simmons JG Liu B Sartor RB Lund PK Growth hormone reduces the severity of fibrosis associated with chronic intestinal inflammation.Gastroenterology. 2005; 129: 204-219Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). For each scoring, observers blinded to the treatment group and genotype examined between 4 and 16 fields in all lung lobes (depending on the size and the homogeneity of the histological changes) using light microscopy (×200 magnification). Fields were examined to cover each entire lobe and were discarded if nonrepresentative areas such as airway lumen occupied >50% of the field of view. The mean score for each lobe was expressed as the average of scores determined in each field. The first scoring system was used to determine the extent of the fibrotic lesions. For each field, the surface of fibrosis was rated as a percentage of the total surface of the field, and the mean percentage (average of percentages determined in each field) reflected the percentage fibrotic area for the lung. Second, the severity of the lesions was determined by using the Ashcroft scoring system18Ashcroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1084) Google Scholar (see Table 118Ashcroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1084) Google Scholar, 19Theiss AL Fuller CR Simmons JG Liu B Sartor RB Lund PK Growth hormone reduces the severity of fibrosis associated with chronic intestinal inflammation.Gastroenterology. 2005; 129: 204-219Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The severity of the fibrotic changes in each lung section was assessed as a mean score of severity from observed microscopic fields. An average of 15 fields within each lung section were observed at a magnification of ×200 in each successive field, and each field was assessed individually for the severity of fibrotic changes and allotted a score from 0 (normal) to 8 (total fibrosis) using a predetermined scale of severity (numerical fibrotic scale). After examination of the whole section, the mean of the scores from all fields was taken as the fibrotic score. To prevent observer bias, all histological specimens were randomly numbered and interpreted in a blinded fashion. Finally, to determine a mean histological score taking in account both the severity and the extent of the fibrotic lesions, a mean score ranging from 0 to 4 to reflect 0%−100% mean fibrotic percentage was assessed for each section, and multiplied by the mean Ashcroft score.Table 1Criteria for Histologic Fibrosis Score of LungScoreDescriptionFibrosis (Ashcroft score, according to Ashcroft et al18Ashcroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1084) Google Scholar)0Normal lung1Minimal fibrous thickening of alveolar or bronchiolar walls2Moderate thickening of walls without obvious damage to lung architecture34Increased fibrosis with definite damage to lung structure and formation of fibrous band or small fibrous masses56Severe distortion of structure and large fibrotic areas, “honeycombing”78Total fibrous obliteration of the fieldPercent involvement00–10% of the section110–25% of section225–50% of section350–75% of section475–100% of sectionAdapted from Theiss et al.19Theiss AL Fuller CR Simmons JG Liu B Sartor RB Lund PK Growth hormone reduces the severity of fibrosis associated with chronic intestinal inflammation.Gastroenterology. 2005; 129: 204-219Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Open table in a new tab Adapted from Theiss et al.19Theiss AL Fuller CR Simmons JG Liu B Sartor RB Lund PK Growth hormone reduces the severity of fibrosis associated with chronic intestinal inflammation.Gastroenterology. 2005; 129: 204-219Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Interleukin-6, monocyte chemotactic protein-1, interferon, and tumor necrosis factor were measured using the BD Cytometric Bead Array Mouse Inflammation Kit (Becton Dickinson, Lincoln Park, NJ) following the manufacturer's instructions. Detection limits were 10 pg/ml. For immunohistochemistry, 4-μm (murine) or 5-μm (human) sections were first deparaffinized and rehydrated. Endogenous peroxidase activity was quenched with 0.3% H2O2 in methanol, and for PAR-1 and PAR-2 staining heat-induced epitope retrieval antigen retrieval was performed using 10 mmol/L citrate buffer [pH 6.0, for 20 minutes. at 98°C in a pretreatment module (Thermo/Labvision, Fremont, CA)]. PAR-1 and PAR-2 stainings were performed with 1:150 anti-PAR-1 H111 (48 hours at 4°C) or 1:200 anti-PAR-2 SAM11 (24 hours at 4°C) antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Smooth muscle actin (aSMA) was visualized using anti-aSMA-1 clone 1A4 (1:500, 1 hour at room temperature, Sigma-Aldrich, St. Louis, MO). A two- or three-step horseradish peroxidase-conjugated polymer detection system (ImmunoLogic, Duiven, The Netherlands) was applied for visualization, using appropriate secondary antibodies and diaminobenzidine staining. Slides were photographed on a Zeiss Axioskop (Zeiss, Jena, Germany) with a Sony DXC950P CCD camera (Sony, Tokyo, Japan). Fibrin and Ly-6 stainings were performed as described previously.20Slofstra SH Cate HT Spek CA Low dose endotoxin priming is accountable for coagulation abnormalities and organ damage observed in the Shwartzman reaction. A comparison between a single-dose endotoxemia model and a double-hit endotoxin-induced Shwartzman reaction.Thromb J. 2006; 4: 13Crossref PubMed Scopus (27) Google Scholar Fibrin staining was graded in a blinded fashion on a 0 to 3 scale in which 0 indicates a lung section with no staining, whereas 1 indicates weak staining, 2 indicates median staining, and 3 indicates intense staining. The number of neutrophils as assessed by LY-6 was scored on 10 microscopic fields at a magnification of ×40. Pulverized lung tissues were lysed in Laemmli lysis buffer, incubated for 5 minutes at 95°C, and whole cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to an immobilon-P polyvinylidene difluoride membrane (Millipore, Billerica, MA). Membranes were incubated overnight at 4°C with primary antibodies against α-actin, β-actin, collagen, PAR-2 (SAM11) (all Santa Cruz, CA), total p42/44 mitogen-activated protein kinase, and epidermal growth factor receptor (Cell Signaling Technology, Beverly, MA). All secondary antibodies were horseradish peroxidase-conjugated from DakoCytomation (Glostrup, Denmark). Blots were imaged using Lumilight Plus ECL substrate from Roche (Basel, Switzerland) on a GeneGnome imager (Syngene, Cambridge, UK). For quantification, densitometry was performed using a GeneTools software (Syngene, Cambridge, UK). Briefly, raw volumes corresponding to the histogram function of the band corresponding to the protein of interest were corrected for those of the loading control (β-actin or epidermal growth factor receptor as indicated). Data are expressed as mean ± SEM of Western blots which were repeated three times. Murine myoblasts C2C12 (CRL-1772), human non-small cell lung cancer cells A549 (CCL-185) and human lung fibroblasts MRC-5 (CCL-171) were purchased from American Type Culture Collection (ATCC), Rockville, MD. Primary murine lung fibroblasts were isolated from wild-type and PAR-2−/− mouse lungs as described in.21Trejo J Connolly AJ Coughlin SR The cloned thrombin receptor is necessary and sufficient for activation of mitogen-activated protein kinase and mitogenesis in mouse lung fibroblasts. Loss of responses in fibroblasts from receptor knockout mice.J Biol Chem. 1996; 271: 21536-21541Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Cells were maintained in Dulbecco's modified Eagle's medium or RPMI (A549 cells) supplemented with 10% fetal calf serum and passed according to routine procedures. Unless stated otherwise, cells were washed twice with PBS, serum-starved for 4 hours and subsequently stimulated as described. A549 cells were used for this assay, as these pulmonary type-II epithelial cell line derived from an individual with alveolar cell carcinoma retained many of the characteristics of normal type-II epithelial cells, such as surfactant production, cytoplasmic multilamellar inclusion bodies, and cuboidal appearance.22Lieber M Smith B Szakal A Nelson-Rees W Todaro G A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells.Int J Cancer. 1976; 17: 62-70Crossref PubMed Scopus (1026) Google Scholar The permeability of A549 monolayers was analyzed in a dual chamber system by measuring the flux of Evans blue-labeled bovine serum albumin (respectively, Sigma, St. Louis, MO, and Calbiochem) across the monolayer as detailed in.23Patterson CE Rhoades RA Garcia JG Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung.J Appl Physiol. 1992; 72: 865-873Crossref PubMed Google Scholar Briefly, A549 cells were plated on Transwell polycarbonate membranes of 0.4-μm pore size (Corning Inc., Corning, NY) and grown to confluence. Subsequently, cells were serum-starved overnight prior stimulation with 100 μmol/L PAR-2 agonist peptide (Anaspec, CA) or PBS as a control. Samples (10 μl) were taken from the lower chamber at the indicated time points and after 10-fold dilution Evans Blue-bovine serum albumin concentrations were measured at 620 nm using a plate reader spectrophotometer. Cells were seeded in 96-well plates in 100 μl of the indicated murine (C2C12 cells, wild-type and PAR-2−/− fibroblasts) or human (MRC-5 cells) BALFs to determine their properties on cell viability. Cell viability was determined at the indicated intervals using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, as described before.13Borensztajn K Stiekema J Nijmeijer S Reitsma PH Peppelenbosch MP Spek CA Factor Xa stimulates proinflammatory and profibrotic responses in fibroblasts via protease-activated receptor-2 activation.Am J Pathol. 2008; 172: 309-320Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Tick anticoagulant peptide (TAP) and hirudin (Calbiochem, San Diego, CA) were used at a concentration of 200 nmol/L (TAP) and 100 nmol/L (hirudin). Statistical analyses were conducted using GraphPad Prism version 3.00 (GraphPad Software, San Diego, CA). The levels in control (saline-treated) animals were set at 100%. Data are expressed as mean ± SEM. Comparisons between two groups were analyzed using two tailed unpaired t-tests when the data where demonstrated to be normally distributed, otherwise Mann-Whitney analysis was performed. P values of <0.05 were considered to indicate a statistically significant difference between two groups. To confirm and expand previous data on the pro-coagulant activity in BALF of IPF patients, we measured thrombin-anti-thrombin complex levels, which are a hallmark of the activation of the coagulation cascade, in the BALF of eight IPF patients. As shown in Figure 1A, BALF thrombin-anti-thrombin complex was increased 55% in IPF patients when compared with healthy volunteers (n = 9) referred to as controls. Furthermore, BALF levels of soluble TF were significantly increased by about 50% in IPF patients compared to controls (Figure 1B). To determine whether coagulation cascade activation in IPF patients was an epiphenomenon, or may play a role in the ongoing progression of fibrosis, we next assessed the functional consequences of human lung fibroblast stimulation with BALF with respect to cell survival. Indeed, both thrombin and FXa have been shown to have a proliferative effect on fibroblasts.6Hernandez-Rodriguez NA Cambrey AD Harrison NK Chambers RC Gray AJ Southcott AM duBois RM Black CM Scully MF McAnulty RJ Laurent GJ Role of thrombin in pulmonary fibrosis.Lancet. 1995; 346: 1071-1073Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 13Borensztajn K Stiekema J Nijmeijer S Reitsma PH Peppelenbosch MP Spek CA Factor Xa stimulates proinflammatory and profibrotic responses in fibroblasts via protease-activated receptor-2 activation.Am J Pathol. 2008; 172: 309-320Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Howell DC Goldsack NR Marshall RP McAnulty RJ Starke R Purdy G Laurent GJ Chambers RC Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2001; 159: 1383-1395Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 24Blanc-Brude OP Archer F Leoni P Derian C Bolsover S Laurent GJ Chambers RC Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation.Exp Cell Res. 2005; 304: 16-27Crossref PubMed Scopus (81) Google Scholar As shown in Figure 1C, BALF of IPF patients enhanced fibroblast survival by 40% as compared to BALF of controls. Strikingly, this increased viability induced by the BALF of IPF patients was significantly reduced by the FXa inhibitor TAP (to 21%), and by the thrombin inhibitor hirudin (to 8%). Incubation of fibroblasts with TAP or hirudin in the absence of BALF did not affect cell survival (data not shown). Lung biopsies of IPF patients showed characteristics of usual interstitial pneumonia, such as the abundant presence of characteristic fibroblast foci. Immunohistochemical staining of lung biopsies from these IPF patients showed prominent PAR-2 expression, especially on smooth muscle cells, but also on epithelial cells overlying a fibroblast focus, and in (myo)fibroblasts of these foci (Figure 2, A and B). In contrast, only sparse staining of alveolar type II cells was observed in the nonfibrotic part of the lung of these patients (Figure 2C). There was no detectable signal for similar sections stained with an isotype-specific control antibody (Figure 2, D and E). To assess the specificity of the PAR-2 antibody, we compared PAR-2 and PAR-1 expression and cellular localization in IPF biopsies. As shown in Supplemental Figure S1 (at http://ajp.amjpathol.org/), fibrotic tissue primarily seemed to show PAR-1 overexpression in epithelial cells in inflamed areas (rather than in epithelial cells overlying fibroblastic foci (Supplemental Figure S1, A and B, at http://ajp.amjpathol.org/) and on macrophages, while in the non fibrotic parts of the lungs, PAR-1 was mainly expressed by type II pneumocytes (Supplemental Figure S1C at http://ajp.amjpathol.org/). Overall, these data suggest that PAR-2 might specifically contribute to the development of pulmonary fibrosis in IPF, support a role for the coag" @default.
• W1541546534 created "2016-06-24" @default.
• W1541546534 creator A5003308528 @default.
• W1541546534 creator A5031445451 @default.
• W1541546534 creator A5033531825 @default.
• W1541546534 creator A5034585119 @default.
• W1541546534 creator A5050478943 @default.
• W1541546534 creator A5055297793 @default.
• W1541546534 creator A5063174626 @default.
• W1541546534 creator A5068095052 @default.
• W1541546534 creator A5069908102 @default.
• W1541546534 creator A5078648606 @default.
• W1541546534 creator A5091250924 @default.
• W1541546534 date "2010-12-01" @default.
• W1541546534 modified "2023-10-09" @default.
• W1541546534 title "Protease-Activated Receptor-2 Induces Myofibroblast Differentiation and Tissue Factor Up-Regulation during Bleomycin-Induced Lung Injury" @default.
• W1541546534 cites W1490750046 @default.
• W1541546534 cites W1531471821 @default.
• W1541546534 cites W17106945 @default.
• W1541546534 cites W172429390 @default.
• W1541546534 cites W1879089985 @default.
• W1541546534 cites W1908907230 @default.
• W1541546534 cites W1918709511 @default.
• W1541546534 cites W1978532495 @default.
• W1541546534 cites W1980278583 @default.
• W1541546534 cites W1980358070 @default.
• W1541546534 cites W1982851659 @default.
• W1541546534 cites W1984119315 @default.
• W1541546534 cites W1991202990 @default.
• W1541546534 cites W1995105009 @default.
• W1541546534 cites W2002856497 @default.
• W1541546534 cites W2006831350 @default.
• W1541546534 cites W2013295033 @default.
• W1541546534 cites W2013650052 @default.
• W1541546534 cites W2014467621 @default.
• W1541546534 cites W2016193552 @default.
• W1541546534 cites W2018000139 @default.
• W1541546534 cites W2018762529 @default.
• W1541546534 cites W2019431738 @default.
• W1541546534 cites W2022178125 @default.
• W1541546534 cites W2027837178 @default.
• W1541546534 cites W2030505427 @default.
• W1541546534 cites W2030898371 @default.
• W1541546534 cites W2031074041 @default.
• W1541546534 cites W2041400348 @default.
• W1541546534 cites W2041404288 @default.
• W1541546534 cites W2042974718 @default.
• W1541546534 cites W2048142530 @default.
• W1541546534 cites W2051772841 @default.
• W1541546534 cites W2051780551 @default.
• W1541546534 cites W2051879911 @default.
• W1541546534 cites W2058278041 @default.
• W1541546534 cites W2058685463 @default.
• W1541546534 cites W2059944938 @default.
• W1541546534 cites W2060126259 @default.
• W1541546534 cites W2068123131 @default.
• W1541546534 cites W2075400164 @default.
• W1541546534 cites W2085809608 @default.
• W1541546534 cites W2089377539 @default.
• W1541546534 cites W2092644867 @default.
• W1541546534 cites W2096879602 @default.
• W1541546534 cites W2101014673 @default.
• W1541546534 cites W2104332008 @default.
• W1541546534 cites W2106210601 @default.
• W1541546534 cites W2110869240 @default.
• W1541546534 cites W2111791469 @default.
• W1541546534 cites W2130104463 @default.
• W1541546534 cites W2134215992 @default.
• W1541546534 cites W2136539876 @default.
• W1541546534 cites W2142531797 @default.
• W1541546534 cites W2143551777 @default.
• W1541546534 cites W2145218316 @default.
• W1541546534 cites W2151998349 @default.
• W1541546534 cites W2154733572 @default.
• W1541546534 cites W2157693596 @default.
• W1541546534 cites W2160041500 @default.
• W1541546534 cites W2161877684 @default.
• W1541546534 cites W2167211373 @default.
• W1541546534 cites W2169712199 @default.
• W1541546534 cites W2171002322 @default.
• W1541546534 cites W2335187016 @default.
• W1541546534 cites W2415371617 @default.
• W1541546534 cites W2416914730 @default.
• W1541546534 doi "https://doi.org/10.2353/ajpath.2010.091107" @default.
• W1541546534 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2993271" @default.
• W1541546534 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20971733" @default.
• W1541546534 hasPublicationYear "2010" @default.
• W1541546534 type Work @default.
• W1541546534 sameAs 1541546534 @default.
• W1541546534 citedByCount "54" @default.
• W1541546534 countsByYear W15415465342012 @default.
• W1541546534 countsByYear W15415465342013 @default.
• W1541546534 countsByYear W15415465342014 @default.
• W1541546534 countsByYear W15415465342015 @default.
• W1541546534 countsByYear W15415465342016 @default.
• W1541546534 countsByYear W15415465342017 @default.
• W1541546534 countsByYear W15415465342018 @default.
• W1541546534 countsByYear W15415465342019 @default.

• next