Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro

Summary

Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1β, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 ± 5.2 % (p<0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1β-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

Keywords

uPA - fibrin - alveolar epithelial repair - pulmonary fibrosis - fibrinolysis

• References

• 1 Loskutoff DJ, Quigley JP. PAI-1, fibrosis, and the elusive provisional fibrin matrix. J Clin Invest 2000; 106: 1441-3.
• 2 Selman M, Pardo A. Idiopathic pulmonary fibrosis: an epithelial/fibroblastic cross-talk disorder. Respir Res 2002; 3: 3.
• 3 Adamson IY, Young L, Bowden DH. Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis. Am J Pathol 1988; 130: 377-83.
• 4 Geiser T. Idiopathic pulmonary fibrosis - a disorder of alveolar wound repair?. Swiss Med Wkly 2003; 133: 405-11.
• 5 Clark RA, Lanigan JM, Della Pelle P. et al Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization. J Invest Dermatol 1982; 79: 264-9.
• 6 Grinnell F. et al Fibroblast adhesion to fibrinogen and fibrin substrata: requirement for cold-insoluble globulin (plasma fibronectin). Cell 1980; 19: 517-25.
• 7 Fuchs-Buder T. et al Time course of procoagulant activity and D dimer in bronchoalveolar fluid of patients at risk for or with acute respiratory distress syndrome. Am J Respir Crit Care Med 1996; 153: 163-7.
• 8 Gunther A, Mosavi P, Heinemann S. et al 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: 454-62.
• 9 Chapman HA, Allen CL, Stone OL. Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease. Am Rev Respir Dis 1986; 133: 437-43.
• 10 Kotani I. et al Increased procoagulant and antifibrinolytic activities in the lungs with idiopathic pulmonary fibrosis. Thromb Res 1995; 77: 493-504.
• 11 Bertozzi P. et al Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990; 322: 890-7.
• 12 Barazzone C, Belin D, Piguet PF. et al Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury. J Clin Invest 1996; 98: 2666-73.
• 13 Pinsky DJ, Liao H, Lawson CA. et al Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition. J Clin Invest 1998; 102: 919-28.
• 14 Idell S. et al Abnormalities of pathways of fibrin turnover in lung lavage of rats with oleic acid and bleomycin-induced lung injury support alveolar fibrin deposition. Am J Pathol 1989; 135: 387-99.
• 15 Sitrin RG. et al Tissue fibrin deposition during acute lung injury in rabbits and its relationship to local expression of procoagulant and fibrinolytic activities. Am Rev Respir Dis 1987; 135: 930-6.
• 16 Eitzman DT, McCoy RD, Zheng X. et al Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest 1996; 97: 232-7.
• 17 Simon RH, Gross TJ, Edwards JA. et al Fibrin degradation by rat pulmonary alveolar epithelial cells. Am J Physiol 1992; 262: L482-8.
• 18 Hasegawa T, Sorensen L, Dohi M. et al Induction of urokinase-type plasminogen activator receptor by IL-1β. Am J Respir Cell Mol Biol 1997; 16: 683-92.
• 19 Hattori N, Sisson TH, Xu Y. et al Upregulation of fibrinolysis by adenovirus-mediated transfer of urokinase-type plasminogen activator genes to lung cells in vitro and in vivo . Hum Gene Ther 1999; 10: 215-22.
• 20 Gross TJ. et al Rat alveolar epithelial cells concomitantly express plasminogen activator inhibitor-1 and urokinase. Am J Physiol 1991; 260: L286-95.
• 21 Geiser T, Jarreau PH, Atabai K. et al Interleukin-1beta augments in vitro alveolar epithelial repair. Am J Physiol Lung Cell Mol Physiol 2000; 279: L1184-90.
• 22 Kiama SG. et al A scanning electron microscope study of the luminal surface specializations in the blood vessels of the pecten oculi in a diurnal bird, the black kite (Milvus migrans). Ann Anat 1998; 180: 455-60.
• 23 Geiser T. et al Pulmonary edema fluid from patients with acute lung injury augments in vitro alveolar epithelial repair by an IL-1β-dependent mechanism. Am J Respir Crit Care Med 2001; 163: 1384-8.
• 24 Pugin J, Ricou B, Steinberg KP. et al Proinflammatory activity in bronchoalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1. Am J Respir Crit Care Med 1996; 153: 1850-6.
• 25 Marshall BC, Xu QP, Rao NV. et al Pulmonary epithelial cell urokinase-type plasminogen activator. Induction by interleukin-1 beta and tumor necrosis factor-alpha. J Biol Chem 1992; 267: 11462-9.
• 26 Spencer H. Interstitial pneumonia. Annu Rev Med 1967; 18: 423-42.
• 27 Idell S. Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Crit Care Med 2003; 31 (Suppl. 04) S213-20.
• 28 Sedo A, Mandys V, Krepela E. Cell membranebound proteases: not “only” proteolysis. Physiol Res 1996; 45: 169-76.
• 29 Hornebeck W. et al Matrix-directed regulation of pericellular proteolysis and tumor progression. Semin Cancer Biol 2002; 12: 231-41.
• 30 Alfano D. et al The urokinase plasminogen activator and its receptor: role in cell growth and apoptosis. Thromb Haemost 2005; 93: 205-11.
• 31 Bass R, Ellis V. Cellular mechanisms regulating non-haemostatic plasmin generation. Biochem Soc Trans 2002; 30: 189-94.
• 32 Lijnen HR. Matrix metalloproteinases and cellular fibrinolytic activity. Biochemistry (Mosc) 2002; 67: 92-8.
• 33 Friedl P, Wolf K. Proteolytic and non-proteolytic migration of tumour cells and leucocytes. Biochem Soc Symp 2003; 277-85.
• 34 Romer J, Bugge TH, Pyke C. et al Impaired wound healing in mice with a disrupted plasminogen gene. Nat Med 1996; 2: 287-92.
• 35 Planus E, Galiacy S, Matthay M. et al Role of collagenase in mediating in vitro alveolar epithelial wound repair. J Cell Sci 1999; 112: 243-52.