Direct Thrombin Inhibition Reduces Lung Collagen, Accumulation, and Connective Tissue Growth Factor mRNA Levels in Bleomycin-Induced Pulmonary Fibrosis
2001; Elsevier BV; Volume: 159; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62525-4
ISSN1525-2191
AutoresDavid C. Howell, Neil Goldsack, Richard P. Marshall, Robin J. McAnulty, Richard Starke, G. Purdy, Geoffrey J. Laurent, Rachel C. Chambers,
Tópico(s)Hemophilia Treatment and Research
ResumoDramatic activation of the coagulation cascade has been extensively documented for pulmonary fibrosis associated with acute and chronic lung injury. In addition to its role in hemostasis, thrombin exerts profibrotic effects via activation of the major thrombin receptor, protease-activated receptor-1. In this study, we examined the effect of the direct thrombin inhibitor, UK-156406 on fibroblast responses in vitro and on bleomycin-induced pulmonary fibrosis in rats. UK-156406 significantly inhibited thrombin-induced fibroblast proliferation, procollagen production, and connective tissue growth factor (CTGF) mRNA levels when used at equimolar concentration to the protease. Thrombin levels in bronchoalveolar lavage fluid and expression of thrombin and protease-activated receptor-1 in lung tissue were increased after intratracheal instillation of bleomycin. The characteristic doubling in lung collagen in bleomycin-treated animals (38.4 ± 2.0 mg versus 17.1 ± 1.4 mg, P < 0.01) was preceded by significant elevations in α1(I) procollagen and CTGF mRNA levels (3.0 ± 0.4-fold and 6.3 ± 0.4-fold respectively, (P < 0.01), and total inflammatory cell number. UK-156406, administered at an anticoagulant dose, attenuated lung collagen accumulation in response to bleomycin by 35 ± 12% (P < 0.05), inhibited α1(I) procollagen and CTGF mRNA levels by 50% and 35%, respectively (P < 0.05), but had no effect on inflammatory cell recruitment. This is the first report showing that direct thrombin inhibition abrogates lung collagen accumulation in bleomycin-induced pulmonary fibrosis. Dramatic activation of the coagulation cascade has been extensively documented for pulmonary fibrosis associated with acute and chronic lung injury. In addition to its role in hemostasis, thrombin exerts profibrotic effects via activation of the major thrombin receptor, protease-activated receptor-1. In this study, we examined the effect of the direct thrombin inhibitor, UK-156406 on fibroblast responses in vitro and on bleomycin-induced pulmonary fibrosis in rats. UK-156406 significantly inhibited thrombin-induced fibroblast proliferation, procollagen production, and connective tissue growth factor (CTGF) mRNA levels when used at equimolar concentration to the protease. Thrombin levels in bronchoalveolar lavage fluid and expression of thrombin and protease-activated receptor-1 in lung tissue were increased after intratracheal instillation of bleomycin. The characteristic doubling in lung collagen in bleomycin-treated animals (38.4 ± 2.0 mg versus 17.1 ± 1.4 mg, P < 0.01) was preceded by significant elevations in α1(I) procollagen and CTGF mRNA levels (3.0 ± 0.4-fold and 6.3 ± 0.4-fold respectively, (P < 0.01), and total inflammatory cell number. UK-156406, administered at an anticoagulant dose, attenuated lung collagen accumulation in response to bleomycin by 35 ± 12% (P < 0.05), inhibited α1(I) procollagen and CTGF mRNA levels by 50% and 35%, respectively (P < 0.05), but had no effect on inflammatory cell recruitment. This is the first report showing that direct thrombin inhibition abrogates lung collagen accumulation in bleomycin-induced pulmonary fibrosis. Pulmonary fibrosis is the end stage of a heterogeneous group of disorders characterized by the excessive deposition of extracellular matrix proteins within the pulmonary interstitium. In addition to increased levels of classical growth factors and cytokines, such as transforming growth factor-β1, platelet-derived growth factor (PDGF), and insulin growth factor-1,1McAnulty RJ Laurent GJ Pathogenesis of lung fibrosis and potential new therapeutic strategies.Exp Nephrol. 1995; 3: 96-107PubMed Google Scholar activation of coagulation cascade with the resultant generation of coagulation proteases has been implicated in the pathogenesis of this disease. Consistent with this observation, intra-alveolar accumulation of fibrin has been described for patients with pulmonary fibrosis,2Chapman HA Allen CL Stone OL Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease.Am Rev Respir Dis. 1986; 133: 437-443PubMed Google Scholar, 3Ikeda T Hirose N Koto H Hirano H Shigematsu N Fibrin deposition and fibrinolysis in the pathogenesis of pulmonary fibrosis.Nippon Kyobu Shikkan Gakkai Zasshi. 1989; 27: 448-451PubMed Google Scholar, 4Kotani I Sato A Hayakawa H Urano T Takada Y Takada A Increased procoagulant and antifibrinolytic activities in the lungs with idiopathic pulmonary fibrosis.Thromb Res. 1995; 77: 493-504Abstract Full Text PDF PubMed Scopus (176) Google Scholar acute lung injury (ALI), and the acute respiratory distress syndrome,5Bachofen M Weibel ER Structural alterations of lung parenchyma in the adult respiratory distress syndrome.Clin Chest Med. 1982; 3: 35-56Abstract Full Text PDF PubMed Google Scholar, 6Idell S Gonzalez K Bradford H MacArthur CK Fein AM Maunder RJ Garcia JG Griffith DE Weiland J Martin TR Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII.Am Rev Respir Dis. 1987; 136: 1466-1474Crossref PubMed Scopus (121) Google Scholar a condition in which rapid fibroproliferation and matrix synthesis can lead to the development of extensive fibrotic lesions.7Marshall RP Bellingan G Laurent GJ The acute respiratory distress syndrome: fibrosis in the fast lane.Thorax. 1998; 53: 815-817Crossref PubMed Scopus (109) Google Scholar Excessive procoagulant activity in the lung is thought to arise from an imbalance in pro- and anti-coagulant factors. For example, bronchoalveolar lavage fluid (BALF) from patients with acute respiratory distress syndrome, has been shown to contain tissue factor/factor VII/VIIa complexes, which can activate factor X, and trigger activation of the coagulation cascade.6Idell S Gonzalez K Bradford H MacArthur CK Fein AM Maunder RJ Garcia JG Griffith DE Weiland J Martin TR Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII.Am Rev Respir Dis. 1987; 136: 1466-1474Crossref PubMed Scopus (121) Google Scholar Increased tissue factor expression on alveolar macrophages and type II pneumocytes, in close association with fibrin deposits, has also been described in the fibrotic lung.8Imokawa 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 (121) Google Scholar We and others have also shown that active thrombin levels are increased in BALF obtained from patients with pulmonary fibrosis associated with systemic sclerosis,9Hernandez-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 (133) Google Scholar, 10Ohba T McDonald JK Silver RM Strange C LeRoy EC Ludwicka A Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor.Am J Respir Cell Mol Biol. 1994; 10: 405-412Crossref PubMed Scopus (115) Google Scholar whereas others have shown that thrombin levels in BALF are elevated in bleomycin-induced pulmonary fibrosis in rats.11Tani K Yasuoka S Ogushi F Asada K Fujisawa K Ozaki T Ogura T Suzuki K The role of thrombin on lung fibroblast growth and fibrosis in bleomycin-induced lung disorder.Nihon Kyobu Shikkan Gakkai Zasshi. 1991; 29: 211-219PubMed Google Scholar, 12Tani K Yasuoka S Ogushi F Asada K Fujisawa K Ozaki T Sano N Ogura T Thrombin enhances lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis.Am J Respir Cell Mol Biol. 1991; 5: 34-40Crossref PubMed Scopus (61) Google Scholar In terms of anticoagulant factors, serum levels of antithrombin III (AT III), which binds and irreversibly inactivates a number of activated serine proteases, including thrombin, are reduced in patients with acute respiratory distress syndrome.13Kirschstein W Heene DL Fibrinolysis inhibition in acute respiratory distress syndrome.Scand J Clin Lab Invest. 1985; 178: S87-S94Google Scholar In addition, decreased activation of protein C, which is the main modulator of coagulation activation, has been shown to be associated with abnormal collagen turnover in the intra-alveolar space of patients with interstitial lung disease.14Yasui H Gabazza EC Taguchi O Risteli J Risteli L Wada H Yuda H Kobayashi T Kobayashi H Suzuki K Adachi Y Decreased protein C activation is associated with abnormal collagen turnover in the intra-alveolar space of patients with interstitial lung disease.Clin Appl Thromb Hemost. 2000; 6: 202-205Crossref PubMed Scopus (31) Google Scholar A number of reports have examined the effects of modulating the coagulation cascade in ALI. For example, exogenous delivery of the highly specific direct thrombin inhibitor hirudin and AT III, have been shown to be protective in animal models of ALI.15Hoffmann H Siebeck M Spannagl M Weis M Geiger R Jochum M Fritz H Effect of recombinant hirudin, a specific inhibitor of thrombin, on endotoxin-induced intravascular coagulation and acute lung injury in pigs.Am Rev Respir Dis. 1990; 142: 782-788Crossref PubMed Scopus (43) Google Scholar, 16Schmidt B Davis P La-Pointe H Monkman S Coates G deSa D Thrombin inhibitors reduce intrapulmonary accumulation of fibrinogen and procoagulant activity of bronchoalveolar lavage fluid during acute lung injury induced by pulmonary overdistention in newborn piglets.Pediatr Res. 1996; 39: 798-804Crossref PubMed Scopus (19) Google Scholar, 17Uchiba M Okajima K Antithrombin III (AT III) prevents LPS-induced pulmonary vascular injury: novel biological activity of AT III.Semin Thromb Hemost. 1997; 23: 583-590Crossref PubMed Scopus (64) Google Scholar In addition, heparin, which inhibits serine proteases by potentiating the formation of AT-III/serine protease complexes, but also has anti-inflammatory properties, lead to improved gas exchange in an animal model of ALI.18Abubakar K Schmidt B Monkman S Webber C deSA D Roberts R Heparin improves gas exchange during experimental acute lung injury in newborn piglets.Am J Respir Crit Care Med. 1998; 158: 1620-1625Crossref PubMed Scopus (24) Google Scholar Finally, heparin has also been shown to attenuate bleomycin-induced pulmonary fibrosis in mice,19Piguet PF Van GY Guo J Heparin attenuates bleomycin but not silica-induced pulmonary fibrosis in mice: possible relationship with involvement of myofibroblasts in bleomycin, and fibroblasts in silica-induced fibrosis.Int J Exp Pathol. 1996; 77: 155-161Crossref PubMed Scopus (27) Google Scholar although it was uncertain in this study whether heparin was delivered at an anticoagulant dose and whether the protective effects were because of the its direct anti-proliferative effects. Thrombin plays a central role in blood coagulation by converting soluble plasma fibrinogen into an insoluble fibrin clot and promoting platelet aggregation and degranulation, but also mediates a number of biological responses that may play important roles in subsequent inflammatory and tissue repair responses. Thrombin activates endothelial cells,20Jaffe EA Grulich J Weksler BB Hampel G Watanabe K Correlation between thrombin-induced prostacyclin production and inositol trisphosphate and cytosolic free calcium levels in cultured human endothelial cells.J Biol Chem. 1987; 262: 8557-8565Abstract Full Text PDF PubMed Google Scholar acts as a chemoattractant for inflammatory cells21Bar-Shavit R Kahn A Fenton II, JW Wilner GD Receptor-mediated chemotactic response of a macrophage cell line (J774) to thrombin.Lab Invest. 1983; 49: 702-707PubMed Google Scholar, 22Bar-Shavit R Kahn A Fenton II, JW Wilner GD Chemotactic response of monocytes to thrombin.J Cell Biol. 1983; 96: 282-285Crossref PubMed Scopus (176) Google Scholar, 23Bizios R Lai L Fenton II, JW Malik AB Thrombin-induced chemotaxis and aggregation of neutrophils.J Cell Physiol. 1986; 128: 485-490Crossref PubMed Scopus (182) Google Scholar and fibroblasts,24Dawes KE Gray AJ Laurent GJ Thrombin stimulates fibroblast chemotaxis and replication.Eur J Cell Sci. 1993; 61: 126-130PubMed Google Scholar and stimulates fibroblast proliferation24Dawes KE Gray AJ Laurent GJ Thrombin stimulates fibroblast chemotaxis and replication.Eur J Cell Sci. 1993; 61: 126-130PubMed Google Scholar, 25Chen LB Buchanan JM Mitogenic activity of blood components. I. Thrombin and prothrombin.Proc Natl Acad Sci USA. 1975; 72: 131-135Crossref PubMed Scopus (415) Google Scholar, 26Carney DH Cunningham DD Cell surface action of thrombin is sufficient to initiate division of chick cells.Cell. 1978; 14: 811-823Abstract Full Text PDF PubMed Scopus (83) Google Scholar and procollagen production.27Chambers RC Dabbagh K McAnulty RJ Gray AJ Blanc-Brude OP Laurent GJ Thrombin stimulates fibroblast pro-collagen production via proteolytic activation of protease-activated receptor 1.Biochem J. 1998; 333: 121-127Crossref PubMed Scopus (140) Google Scholar Most of the cellular effects of thrombin are mediated by a unique family of ubiquitously expressed cell-surface receptors called protease-activated receptors (PARs), which are activated by limited proteolysis rather than direct ligand binding.28Vu TK Hung DT Wheaton VI Coughlin SR Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2828) Google Scholar To date, four PARs have been characterized, of which three, (PAR-1, -3, and -4), are activated by thrombin, although PAR-1 has been shown to be the major receptor involved in mediating thrombin's mitogenic, profibrotic, and proinflammatory effects in vitro.27Chambers RC Dabbagh K McAnulty RJ Gray AJ Blanc-Brude OP Laurent GJ Thrombin stimulates fibroblast pro-collagen production via proteolytic activation of protease-activated receptor 1.Biochem J. 1998; 333: 121-127Crossref PubMed Scopus (140) Google Scholar, 29Trejo 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, 30Cunningham MA Rondeau E Chen X Coughlin SR Holdsworth SR Tipping PG Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis.J Exp Med. 2000; 191: 455-462Crossref PubMed Scopus (190) Google Scholar After the interaction of thrombin with its receptors, most of its cellular effects are thought to be mediated via the induction and release of a host of secondary mediators.31Dery O, Corvera C, Steinhoff M, Bunnet N: Proteinase-activated receptors: novel mechanisms of signalling by serine proteases. Am J Physiol, 274:C1429--C1452Google Scholar For example, there is good evidence that the mitogenic effects of thrombin for lung fibroblasts in vitro are mediated, at least in part via the autocrine production of PDGF-AA and up-regulation of the PDGF-α receptor.10Ohba T McDonald JK Silver RM Strange C LeRoy EC Ludwicka A Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor.Am J Respir Cell Mol Biol. 1994; 10: 405-412Crossref PubMed Scopus (115) Google Scholar More recently, we have shown that thrombin is also a potent inducer of connective tissue growth factor (CTGF) production by human lung fibroblasts via direct proteolytic activation of PAR-1.32Chambers R Leoni P Blanc-Brude O Wembridge D Laurent G Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1.J Biol Chem. 2000; 275: 35584-35591Crossref PubMed Scopus (214) Google Scholar CTGF is a fibroblast mitogen, chemoattractant, and promoter of procollagen and fibronectin production in vitro.33Frazier K Williams S Kothapalli D Klapper H Grotendorst GR Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor.J Invest Dermatol. 1996; 107: 404-411Crossref PubMed Scopus (680) Google Scholar It has therefore been proposed that some of the profibrotic effects of thrombin in vitro may also be mediated, at least in part, by increased production of this growth factor. CTGF is also induced in response to transforming growth factor-β1 and is thought to be responsible for mediating some of its downstream fibrogenic effects.34Grotendorst GR Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts.Cytokine Growth Factor Rev. 1997; 8: 171-179Abstract Full Text PDF PubMed Scopus (681) Google Scholar Further evidence that CTGF may be important in tissue repair and fibrosis has been provided by studies showing that repeated subcutaneous injection of CTGF into newborn mice leads to increased connective tissue deposition,33Frazier K Williams S Kothapalli D Klapper H Grotendorst GR Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor.J Invest Dermatol. 1996; 107: 404-411Crossref PubMed Scopus (680) Google Scholar and that CTGF expression is dramatically increased in a number of fibrotic and fibroproliferative disorders, including pulmonary fibrosis35Allen JT Knight RA Bloor CA Spiteri MA Enhanced insulin-like growth factor binding protein-related protein 2 (connective tissue growth factor) expression in patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis.Am J Respir Cell Mol Biol. 1999; 21: 693-700Crossref PubMed Scopus (169) Google Scholar and in animal models of this disease.36Lasky JA Ortiz LA Tonthat B Hoyle GW Corti M Athas G Lungarella G Brody A Friedman M Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis.Am J Physiol. 1998; 275: L365-L371PubMed Google Scholar Despite the evidence that thrombin may play an important role in the pathogenesis of pulmonary fibrosis, there have been no previous reports that have specifically addressed whether direct thrombin inhibition affects collagen accumulation in this disorder. The aim of this study was therefore to examine the effect of a potent and highly selective direct thrombin inhibitor, UK-156406 (Pfizer Central Research, Sandwich, Kent, UK) on thrombin-induced fibroblast responses in vitro and in bleomycin-induced pulmonary fibrosis in rats. Our data show that UK-156406 blocked the profibrotic effects of thrombin in vitro when used at equimolar concentration to the protease and attenuated lung collagen accumulation after bleomycin-induced lung injury. We further show that the protective effect of direct thrombin inhibition on lung collagen accumulation in this model was preceded by significant reductions in both α1(I) procollagen and CTGF mRNA levels, but not by changes in inflammatory cell recruitment. This is, to our knowledge, the first report to show that direct thrombin inhibition is associated with attenuation of both α1(I) procollagen and CTGF mRNA levels, and ultimately an abrogation in lung collagen accumulation in an animal model of pulmonary fibrosis. Purified human α-thrombin (catalog no.T4393) was obtained from Sigma Chemical Co. Ltd., (Poole, Dorset, UK). The direct thrombin inhibitor, UK-156406 was a generous gift from Dr. Andrew Gray (Pfizer Central Research, Sandwich, Kent, UK). The cDNA probe for human CTGF was inserted into the Eco RI and Not I sites of pBluescript and kindly provided by Dr. Raj Beri (AstraZeneca R&D Charnwood, Loughborough, UK). The cDNA probe for FISP12 (the murine orthologue of CTGF), encompassing nucleotides 1663 to 2930 was generated from a plasmid (pBluescriptfisp12del) kindly provided by Dr. Joseph A. Lasky (Tulane University, New Orleans, LA). The pBluescriptfisp12del plasmid was subcloned from a plasmid (A12/pMexNeo I) originally obtained from Dr. Rolf-Peter Ryseck (Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). The cDNA probe for α1(I) procollagen (probe Hf677) was kindly provided by Dr. M. L. Chu (Thomas Jefferson University, Philadelphia, PA). Rat-specific PAR-1 antibodies were generated by immunizing rabbits with the agonist sequence SFFLRNPSENTFELVPL-NH2 and purified by affinity chromatography37Jenkins AL Bootman MD Taylor CW Mackie EJ Stone SR Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells.J Biol Chem. 1993; 268: 21432-21437Abstract Full Text PDF PubMed Google Scholar and were a generous gift from Professor Eleanor Mackie (University of Melbourne, Melbourne, Australia). Human fetal lung fibroblasts (HFL-1; American Type Culture Collection, Rockville, MD), were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with penicillin, streptomycin, and 10% (v/v) newborn calf serum (NCS), (Imperial Laboratories, Andover, Hampshire, UK). Cells were routinely passaged, tested for mycoplasma infection, and used for experiments between passages l5 to 20. In all in vitro experiments, UK-156406 was preincubated with human α-thrombin (10 nmol/L to 1 μmol/L) in serum-free DMEM for 60 minutes at 37°C. To examine the effects of thrombin alone, the protease was similarly preincubated in serum-free DMEM under the same conditions. Male Lewis rats, aged 6 weeks and weighing 140 to 170 g, were anesthetized by intramuscular injection of 0.75 to 1.0 ml/kg Hypnorm (fentanyl citrate, 0.315 mg/ml, and fluanisone, 10 mg/ml; Janssen Pharmaceutical, High Wycombe, UK). Bleomycin sulfate (Lundbeck, Luton, UK) was administered by a single intratracheal injection (l.5 mg/kg body weight in 0.3 ml of sterile saline) as described previously.38Mutsaers SE Foster ML Chambers RC Laurent GJ McAnulty RJ Increased endothelin-1 and its localization during the development of bleomycin-induced pulmonary fibrosis in rats.Am J Respir Cell Mol Biol. 1998; 18: 611-619Crossref PubMed Scopus (92) Google Scholar Control animals received 0.3 ml of saline alone. In initial experiments, groups of six rats were killed by pentobarbitone overdose after 6 days to allow assessment of thrombin levels in BALF, as described previously.39McAnulty RJ Guerreiro D Cambrey AD Laurent GJ Growth factor activity in the lung during compensatory growth after pneumonectomy: evidence of a role for IGF-1.Eur Respir J. 1992; 5: 739-747PubMed Google Scholar Separate groups of two rats were sacrificed 1, 3, 6, and 14 days after bleomycin instillation for immunohistochemical assessment of thrombin and PAR-1. Lungs were fixed by intratracheal instillation of 4% paraformaldehyde, the trachea ligated, and the inflated lungs and heart removed en bloc. Tissues were fixed and transferred to 15% sucrose in phosphate-buffered saline (PBS), before alcohol dehydration and embedding in paraffin wax. For assessment of the effect of UK-156406 on lung collagen accumulation after bleomycin-induced lung injury at 14 days, UK-156406 (0.5 mg/kg/hour in 0.9% sterile saline) was administered continuously via osmotic minipumps (Alzet, Palo Alto, CA) implanted subcutaneously, 24 hours before bleomycin instillation, to groups of six animals. Drug control animals received minipumps containing saline alone. An additional series of animals was killed 6 days after bleomycin or saline instillation for measurement of blood coagulation parameters, total and differential cell counts in BALF, and for Northern analysis of lung tissue CTGF and α1(I) procollagen mRNA levels. For measurement of coagulation parameters, blood was collected from the inferior vena cava of animals after laparotomy, and was immediately mixed, 10:1 with a solution of 3.8% trisodium citrate (w/v). For measurement of total lung collagen and CTGF and procollagen mRNA levels, the vasculature was perfused with 5 ml of sterile saline containing 100 U/ml heparin. The lungs were removed, weighed, and immediately snap-frozen in liquid N2 after removing the trachea and major airways. Fibroblast proliferation was assessed using a colorimetric assay based on the uptake and subsequent elution of the dye methylene blue as previously described.40Oliver MH Harrison NK Bishop JE Cole PJ Laurent GJ A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors.J Cell Sci. 1989; 92: 513-518Crossref PubMed Google Scholar Briefly, cells were seeded at 5 × 103 cells/well into 96-well plates (Nunc, Life Technologies, Paisley, Scotland, UK) in DMEM and 5% NCS. Control medium, thrombin, or thrombin plus UK-156406 (10 nmol/L to 1 μmol/L) were added to cell cultures for 48 hours. Cells were rinsed with PBS, fixed with formol-saline, and stained with a solution of methylene blue for 30 minutes. Plates were rinsed with borate buffer, bound dye was eluted from the cell monolayer by addition of acidified alcohol, and the absorbance was measured at 650 nmol/L using a microplate spectrophotometer. In some experiments, changes in fibroblast cell number were confirmed by direct cell counting with a standard hemocytometer (British Drug House/Merck, UK). Fibroblast procollagen production in vitro and total lung collagen in vivo were assessed by quantitating hydroxyproline by reverse-phase HPLC as previously described.38Mutsaers SE Foster ML Chambers RC Laurent GJ McAnulty RJ Increased endothelin-1 and its localization during the development of bleomycin-induced pulmonary fibrosis in rats.Am J Respir Cell Mol Biol. 1998; 18: 611-619Crossref PubMed Scopus (92) Google Scholar, 41Campa JS McAnulty RJ Laurent GJ Application of high-pressure liquid chromatography to studies of collagen production by isolated cells in culture.Anal Biochem. 1990; 186: 257-263Crossref PubMed Scopus (55) Google Scholar, 42Chambers RC McAnulty RJ Shock A Campa JS Newman Taylor AJ Laurent GJ Cadmium selectively inhibits fibroblast procollagen production and proliferation.Am J Physiol. 1994; 267: L300-L308PubMed Google Scholar Briefly, for in vitro studies, cells were seeded at 105 cells/ml in 2.4-cm diameter wells in DMEM and 5% NCS, grown to visual confluence and exposed to either control medium, thrombin, or thrombin plus with UK-156406 (10 nmol/L to 1 μmol/L) for 48 hours. At the end of the incubation period, proteins in the media and cell layer were ethanol precipitated and separated from free amino acids by filtration (0.45 μmol/L). Filters were hydrolyzed in HCl and hydrolysates prepared for quantitation of hydroxyproline by HPLC analysis (Beckman System Gold, Beckman, High Wycombe, UK), after derivatization with 7-chloro-4-nitrobenzofuran (Sigma) as previously described.42Chambers RC McAnulty RJ Shock A Campa JS Newman Taylor AJ Laurent GJ Cadmium selectively inhibits fibroblast procollagen production and proliferation.Am J Physiol. 1994; 267: L300-L308PubMed Google Scholar For measurement of total lung collagen, powdered lung tissue was weighed and was similarly hydrolyzed in HCl and prepared for HPLC analysis after diluting hydrolysate aliquots (1:100). The total amount of collagen in each lung was calculated, assuming that lung collagen contains 12.2% w/w hydroxyproline43Laurent GJ Cockerill P McAnulty RJ Hastings JR A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples.Anal Biochem. 1981; 113: 301-312Crossref PubMed Scopus (113) Google Scholar and results were expressed as total lung collagen (mg). For Northern analysis of CTGF mRNA levels in vitro, fibroblasts were seeded at 2 × 105 cells/ml in 6-cm diameter dishes in DMEM and 5% NCS as described previously.32Chambers R Leoni P Blanc-Brude O Wembridge D Laurent G Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1.J Biol Chem. 2000; 275: 35584-35591Crossref PubMed Scopus (214) Google Scholar Briefly, on reaching visual confluence, cells were quiesced for 24 hours and exposed to control media, thrombin, or thrombin plus with UK-156406, (10 nmol/L to 100 nmol/L) in serum-free conditions. After 90 minutes, total RNA was isolated with TRIzol reagent according to the manufacturer's instructions (Gibco BRL, Paisley, UK). For Northern analysis of CTGF and α1(I) procollagen mRNA levels in vivo, total RNA was isolated with TRIzol reagent, from powdered lung tissue, kept frozen. Total RNA from fibroblast cultures (5 μg), and lung tissue (10 μg) were mixed with RNA loading buffer (Sigma), and electrophoresed on a formaldehyde 1% (w/v) agarose gel. RNA loading and integrity was visualized and quantitated by fluorescent scanning of the gel (Fuji, FLA 3000) before transfer to nylon membranes (Hybond N; Amersham International, High Wycombe, UK) and fixation by UV crosslinking. Membranes were hybridized overnight in Denhardt's-based standard hybridization solution at 65°C in the presence of the [32P]-dCTP-labeled cDNA probes for human CTGF (fibroblast cultures), or α1(I) procollagen and FISP12 (lung tissue). At the end of the hybridization, membranes probed for CTGF and FISP12 were rinsed at low stringency [2× standard saline citrate (SSC), 0.1% sodium dodecyl sulfate (SDS), for 5 minutes at room temperature], once at medium stringency (0.5× SSC, 0.1% SDS, for 25 minutes at 65°C) and once at high stringency (0.1× SSC, 0.1% SDS, for 5 minutes at 65°C). Membranes probed for α1(I) procollagen were rinsed at low stringency (2× SSC, 0.1% SDS, 5 minutes at room temperature, followed by 20 minutes at 65°C) and high
Referência(s)