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. 2000 Dec;106(11):1341-50.
doi: 10.1172/JCI10531.

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice

N Hattori  1 , J L Degen, T H Sisson, H Liu, B B Moore, R G Pandrangi, R H Simon, A F Drew
Affiliations

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice

N Hattori et al. J Clin Invest. 2000 Dec.

Abstract

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

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Figures

Figure 1
Figure 1
Effect of bleomycin on lung hydroxyproline content and survival in PAI-1–/– and PAI-1+/+ mice. Bleomycin (2.5 U/kg) or PBS was instilled intratracheally into PAI-1–/– and PAI-1+/+ mice. (a) On days 14, 21, and 28 after administration, lung hydroxyproline was measured. Data are expressed as mean ± SEM; n = 9–11 mice per group. (b) Kaplan-Meier survival curve for bleomycin-exposed PAI-1–/– and PAI-1+/+ mice (n = 20 per group). Bleo, bleomycin.
Figure 2
Figure 2
Dynamics of fibrin accumulation in bleomycin-exposed mice. Bleomycin (2.5 U/kg) or PBS was instilled intratracheally into PAI-1–/– and PAI-1+/+ mice. (a) To measure tissue fibrin content, lungs were harvested from heparinized mice on the days indicated and were processed as described in Methods. As positive and negative controls, fibrin samples formed within thrombin-treated plasma and anticoagulated plasma were prepared and processed in an identical manner. Samples were separated by SDS-PAGE and then immunoblotted to detect plasmin-generated, fibrin-derived γ-γ dimer fragment. (b) Vascular permeability was measured 7 days and 14 days after bleomycin administration, using lung accumulation of Evans blue dye that was injected intravenously 1 hour before sacrifice. Data are expressed as mean ± SEM; n = 7 for bleomycin-injured mice, n = 3 for PBS control mice. (c) The rate of fibrin degradation was measured in lungs of PAI-1–/– and PAI-1+/+ mice 7 days and 14 days after intratracheal instillation of PBS or bleomycin. At the time of sacrifice, fibrin was formed within pulmonary airspaces by intratracheally instilling fluorescein-labeled fibrinogen, plasminogen, and thrombin. After 5 hours, the percent of soluble fluorescent material was measured. Data are expressed as mean ± SEM; n = 4–7 mice per group.
Figure 3
Figure 3
Effects of tranexamic acid on lungs of bleomycin-injured PAI-1–/– mice. PBS or bleomycin was instilled intratracheally into PAI-1–/– mice, and PBS or tranexamic acid was administered by subcutaneous osmotic pump and addition to drinking water. After 14 days, lungs were harvested and analyzed. (a) Immunoblot of fibrin-derived γ-γ dimer fragment in the plasmin-digested lungs of tranexamic acid–treated PAI-1–/– mice. (b) Hydroxyproline content of lung tissue. Results are expressed as mean ± SEM; n = 5–8 mice per bleomycin-treated group.
Figure 4
Figure 4
Effect of bleomycin (5 U/kg) on lung hydroxyproline content of fib–/– and fib+/– mice. Bleomycin (fib+/–, n = 6; fib–/–, n = 5) or PBS (fib+/–, n = 6; fib–/–, n = 3) was instilled intratracheally into fib–/– and fib+/– mice. Twenty-four days after administration, lung hydroxyproline was measured. Data are expressed as mean ± SEM.
Figure 5
Figure 5
Microscopic appearance of focal fibrotic lesions in lungs of fib+/– and fib–/– mice treated with bleomycin. Fibrotic lesions with associated inflammatory infiltrates typical of those occurring in lungs of fib+/– (a) and fib–/– (b) mice 24 days after bleomycin instillation (hematoxylin and eosin staining). Gomori’s trichrome stain illustrating collagen deposition within lesions of both fib+/– (c) and fib–/– (d) mice. Immunohistochemical stain showing that fibrin(ogen) is associated with lesions of fib+/– mice (e; brown reaction product) but is absent in fib–/– mice (f). Immunostaining for fibronectin shows a similar pattern in fib+/– (g) and fib–/– (h) mice.
Figure 6
Figure 6
Effect of tranexamic acid on lung hydroxyproline content of bleomycin-treated lungs of fib+/– and fib–/– mice. PBS or bleomycin was instilled intratracheally, and PBS or tranexamic acid was administered by subcutaneous osmotic pump and addition to drinking water. After 14 days, lungs were harvested and analyzed for hydroxyproline content. Results expressed as mean ± SEM; n = 4–7 animals per group. Tran, tranexamic acid.
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