The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system

Abstract

Acute and chronic pulmonary diseases are characterized by impaired fibrinolytic activity within the lung. To determine the role of the fibrinolytic system in regulating the pathologies associated with lung injury, we examined the effect of bleomycin, an agent that induces the development of pulmonary fibrosis, in mice deficient for plasminogen (Pg(-)(/-)), urokinase (u-PA(-)(/-)), urokinase receptor (u-PAR(-)(/-)), or tissue plasminogen activator (t-PA(-)(/-)), and in control wild-type (WT) mice. Pg(-)(/-) and t-PA(-)(/-) mice demonstrated an enhanced increase in lung collagen content relative to that observed in WT mice. Levels in u-PA(-)(/-) and u-PAR(-)(/-) mice were similar to those in WT mice. Histological analysis 14 days after lung injury confirmed enhanced interstitial fibrosis in Pg(-)(/-), u-PA(-)(/-), and t-PA(-)(/-) mice relative to WT and u-PAR(-)(/-) mice. Areas of pulmonary hemorrhage were observed in bleomycin-treated WT mice and not in Pg(-)(/-), u-PA(-)(/-), and u-PAR(-)(/-) mice or saline controls. Instead, extensive areas of fibrosis were present throughout the lungs of bleomycin-treated Pg(-)(/-) and u-PA(-)(/-) mice. A mixed phenotype (hemorrhage and fibrosis) was observed in t-PA(-)(/-) and Pg(+/-) mice. Hemosiderin-laden macrophages were abundant in the lungs of mice exhibiting hemorrhage and these mice were prone to an early death. Enhanced macrophage levels in the lungs and activation of matrix metalloelastase (MMP-12) were found in mice with a hemorrhage phenotype. The results of these studies indicate a role for the fibrinolytic system in acute lung injury and suggests that intra-alveolar hemorrhage is the result of basement membrane degradation through cell-mediated u-PA activation of Pg with possible involvement of matrix metalloproteinases. Absence of these two components of the fibrinolytic system, either urokinase or plasminogen, results in accelerated fibrosis.

Figure 1.

Hydroxyproline content in lungs 14 days after saline (n = 3 for each genotype) or bleomycin treatment of WT (n = 4), Pg^+/− (n = 5), Pg^−/− (n = 5), u-PA^−/− (n = 5), u-PAR^−/− (n = 7), and t-PA^−/− (n = 4) mice. Values are means ± SE of 4 to 7 mice, performed in duplicate.

Figure 2.

Photomicrographs of representative lungs from WT (n = 6; A−D), Pg^−/− (n = 7; E and F), and Pg^+/− (n = 7; G and H) mice 14 days after saline or bleomycin treatment. A−C and E−G were stained with Masson’s trichrome; D and H were stained with Prussian blue. A: Saline-treated WT lung showing a bronchiole (b) and artery (a) from the bronchioarterial bundle surrounded by normal deposition of collagen (arrow) and normal alveoli; original magnification, ×100. B: Bleomycin-treated WT lung showing focal areas of fibrosis (f) and proteinaceous exudate (arrows) in the alveolar spaces; original magnification, ×100. C: Another area in the lung in B, showing extensive intra-alveolar hemorrhage (RBCs) and fibrin deposition (arrow); original magnification, ×100. D: Adjacent section to the lung in C showing hemosiderin-laden macrophages (arrows) associated to the area of extensive alveolar hemorrhage; original magnification, ×100. E: Bleomycin-treated Pg^−/− lung showing extensive fibrosis (f). No intra-alveolar hemorrhage was observed in the lungs of these animals; original magnification, ×100. F: Higher magnification of the lung in E, showing extensive deposition of collagen (arrows) associated with fibrotic areas (f); original magnification, ×400. G: Bleomycin-treated Pg^+/− lung showing both areas of intra-alveolar hemorrhage (h) and fibrosis (f); original magnification, ×100. H: Adjacent section to the lung in G, showing hemosiderin-laden macrophages (arrows) associated with areas of extensive hemorrhage (h) but not with fibrotic areas (f); original magnification, ×100.

Figure 3.

Photomicrographs of representative lungs (n = 6 for each genotype) from mice 14 days after bleomycin treatment stained with Masson’s trichrome. A: u-PA^−/− lung showing extensive area of fibrosis (f); original magnification, ×100. B: u-PAR^−/− lung showing focal areas of fibrosis (f) surrounded by normal alveoli; original magnification, ×100. C: t-PA^−/− lung showing areas of fibrosis (f) surrounded by areas of extensive intra-alveolar hemorrhage (h); original magnification, ×100.

Figure 4.

Photomicrographs of representative mice lungs (n = 3 for each genotype) treated with bleomycin and immunostained with an anti-fibrin (ogen) antibody. A: WT lung 14 days after treatment showing few areas of fibrin deposition (arrows); original magnification, ×100. B: Pg^−/− lung 7 days after treatment showing extensive fibrin deposition (brown aggregates); original magnification, ×40. C: Pg^−/− lung 14 days after treatment showing extensive fibrin deposition (arrows) associated with areas of fibrosis (f); original magnification, ×200.

Figure 5.

Western blot showing MMP-12 expression in representative whole lung extracts from WT (n = 3/time point) and Pg^−/− (n = 3/time point) mice 5 and 14 days after bleomycin treatment. Relative molecular weights are shown on the left.

Figure 6.

Photomicrographs of representative sections of lungs from bleomycin-treated WT mice (n = 3) 14 days after treatment immunostained with an anti-MMP-12 antibody. A: Deposition of MMP-12 is observed in the alveolar walls (arrows) associated with areas of extensive hemorrhage (h); original magnification, ×100. B: MMP-12 is concentrated in the capillaries (arrows) in the wall of the alveoli (a); original magnification, ×400.

Figure 7.

Proposed model for the role of the fibrinolytic system in acute lung injury.