The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism

Abstract

Mice deficient in the plasminogen activator inhibitor-1 gene (PAI-1-/- mice) are relatively protected from developing pulmonary fibrosis from bleomycin administration. We hypothesized that one of the protective mechanisms may be the ability of the plasminogen system to enhance hepatocyte growth factor (HGF) effects, which have been reported to be anti-fibrotic in the lung. HGF is known to be sequestered in tissues by binding to extracellular matrix components. Following bleomycin administration, we found that HGF protein levels were higher in bronchoalveolar lavage fluid from PAI-1-/- mice compared to wild-type (PAI-1+/+) mice. This increase could be suppressed by administering tranexamic acid, which inhibits plasmin activity. Conversely, intratracheal instillation of urokinase into bleomycin-injured PAI-1+/+ mice to activate plasminogen caused a significant increase in HGF within bronchoalveolar lavage and caused less collagen accumulation in the lungs. Administration of an anti-HGF neutralizing antibody markedly increased collagen accumulation in the lungs of bleomycin-injured PAI-1-/- mice. These results support the hypothesis that increasing the availability of HGF, possibly by enhancing its release from extracellular matrix by a plasmin-dependent mechanism, is an important means by which activation of the plasminogen system can limit pulmonary fibrosis.

Figure 1

Expression of HGF mRNA and protein in the lungs of bleomycin-injured mice. Bleomycin or PBS was instilled intratracheally into PAI-1^−/− and PAI-1^+/+ mice. After 7 days, lungs were harvested and the vasculature perfused with PBS. a: Total RNA was extracted from the lungs and examined for HGF and β-actin mRNA by semiquantitative RT-PCR. b: Optical density of each PCR product was measured and the HGF mRNA expression level expressed as its ratio to the density of the β-actin band (* P < 0.01 for bleomycin PAI-1^−/− mice versus PBS PAI-1^−/− mice, * P > 0.5 for bleomycin PAI-1^−/− mice versus bleomycin PAI-1^+/+ mice, ** P < 0.01 for bleomycin PAI-1^+/+ mice versus PBS PAI-1^+/+ mice). c: Tissue extracts from lungs were prepared as described in Materials and Methods. HGF level in the extract was measured by ELISA. Data are expressed as mean ± SEM; n = 3 to 4 mice per group (* P < 0.01 for bleomycin PAI-1^−/− mice versus PBS PAI-1^−/− mice, * P > 0.5 for bleomycin PAI-1^−/− mice versus bleomycin PAI-1^+/+ mice, ** P < 0.01 for bleomycin PAI-1^+/+ mice versus PBS PAI-1^+/+ mice).

Figure 2

HGF levels in BAL fluids. Bleomycin or PBS was instilled intratracheally into PAI-1^−/− and PAI-1^+/+ mice. BAL fluid was obtained 7 days after bleomycin or PBS administration and HGF level was measured by ELISA. Tranexamic acid treatment was performed as described in Methods. Data are expressed as mean ± SEM; n = 4 to 6 mice per group (* P < 0.01 for bleomycin/tranexamic acid PAI-1^−/− mice versus bleomycin PAI-1^+/+ mice; ** P < 0.05 for bleomycin/tranexamic acid PAI-1^−/− mice versus bleomycin/tranexamic acid PAI-1^+/+).

Figure 3

Molecular forms of HGF in BAL fluid and lung tissue. Bleomycin or PBS was instilled intratracheally into PAI-1^−/− and PAI-1^+/+ mice. The heparin-binding fraction was prepared from BAL fluid and lung homogenate obtained 7 days after bleomycin administration. After dialyzing against PBS, this fraction was concentrated and the HGF level was determined by ELISA. Approximately 100 pg of HGF in each concentrated fraction was subjected to SDS-PAGE under reducing condition and immunoblotted with an anti-HGF α-chain antibody. Bands corresponding in molecular weights to the single chain of HGF (97 kd), the α-chain of HGF (69 kd), and a non-glycosylated α-chain (54 kd) were detected.

Figure 4

Effect of neutralizing antibody against HGF on lung collagen accumulation in bleomycin-injured PAI-1^−/− mice. Bleomycin was instilled intratracheally into PAI-1^−/− mice, and anti-rat HGF rabbit serum (αHGF IgG) or normal rabbit serum (control IgG) was administered intraperitoneally as described in Materials and Methods. After 14 days, lungs were harvested and analyzed for collagen content using the Sircol biochemical assay. To evaluate baseline collagen content in lungs, lungs from PAI-1^−/− mice without any treatment were also assayed. Data are expressed as mean ± SEM; n = 5 to 6 mice per group (* P < 0.01 αHGF versus control IgG).

Figure 5

Effect of urokinase on HGF level in BAL fluid and lung collagen accumulation in bleomycin-injured PAI-1^+/+ mice. Bleomycin or PBS was instilled intratracheally into PAI-1^+/+ mice. On day 6 after administration, urokinase or heat-inactivated urokinase was injected via exposed trachea. a: On day 7 after bleomycin treatment, BAL fluid was obtained and analyzed for HGF level by ELISA (n = 4 to 5 mice per group) (* P < 0.05 urokinase versus inactivated urokinase). b: The remaining mice (n = 6 to 8 mice per group) were sacrificed on day 14 after bleomycin administration to determine collagen content in the lungs. Data are expressed as mean ± SEM (* P < 0.05 for urokinase versus inactivated urokinase).