Hematopoietic protease nexin-1 protects against lung injury by preventing thrombin signaling in mice

Abstract

Coagulation and fibrinolytic system deregulation has been implicated in the development of idiopathic pulmonary fibrosis, a devastating form of interstitial lung disease. We used intratracheal instillation of bleomycin to induce pulmonary fibrosis in mice and analyzed the role of serine protease inhibitor E2 (serpinE2)/protease nexin-1 (PN-1), a tissue serpin that exhibits anticoagulant and antifibrinolytic properties. PN-1 deficiency was associated, after bleomycin challenge, with a significant increase in mortality, as well as a marked increase in active thrombin in bronchoalveolar lavage fluids, an overexpression of extracellular matrix proteins, and an accumulation of inflammatory cells in the lungs. Bone marrow transplantation experiments showed that protective PN-1 was derived from hematopoietic cell compartment. A pharmacological strategy using the direct thrombin inhibitor argatroban reversed the deleterious effects of PN-1 deficiency. Concomitant deficiency of the thrombin receptor protease-activated receptor 4 (PAR4) abolished the deleterious effects of PN-1 deficiency in hematopoietic cells. These data demonstrate that prevention of thrombin signaling by PN-1 constitutes an important endogenous mechanism of protection against lung fibrosis and associated mortality. Our findings suggest that appropriate doses of thrombin inhibitors or PAR4 antagonists may provide benefit against progressive lung fibrosis with evidence of deregulated thrombin activity.

Graphical abstract

Figure 1.

Increased mortality in bleomycin-injured PN-1^−/−mice. PN-1^−/− mice and their PN-1^+/+ littermates were subjected to bleomycin-induced lung injury (2 mg/kg) by intratracheal instillation. Percentages of surviving mice were plotted over a 14-day period after bleomycin treatment. Log-rank test was used to compare the difference between PN-1^−/− and PN-1^+/+ mice (n = 25 per group; P ≤ .0001). The proportion of survival was determined based on euthanasia criteria. Animals that lost 20% of their body weight were euthanized.

Figure 2.

Accentuated pulmonary inflammatory and coagulation responses in lungs of bleomycin-injured PN-1^−/−mice. PN-1^−/− and their PN-1^+/+ littermates were subjected to bleomycin-induced lung injury (BLM; 2 mg/kg) or physiological serum (sham) by intratracheal instillation for 4 to 5 days. BALFs were collected from lung tissues and the number of WBCs (A), platelets (B), and red blood cells (RBCs) (C) were determined by counting in the hemocytometer. Thrombin activity was measured by the fluorometric method (D) and D-dimer by enzyme-linked immunosorbent assay (E). Data (mean ± standard error of the mean [SEM]; n = 6-8 per group) were analyzed by 1-way analysis of variance with a Tukey’s multiple comparison test. *P < .05, **P < .01, ***P < .001, ****P < .0001 vs respective control. ns, not significant.

Figure 3.

Accentuated pulmonary fibrosis and increased TGFβ1 in PN-1^−/−mice. Lungs from PN-1^−/− mice and their PN-1^+/+ littermates were harvested at the indicated time points after bleomycin-induced lung injury (BLM) instillation for the following analyses. (A) Masson’s trichrome (i-iv, ix-xii) and Sirius red (v-viii, xiii-xvi) staining of lung sections from PN-1^+/+ and PN-1^−/− mice 3 and 9 days after saline (sham) or BLM treatment. Representative images are shown. Scale bars, 1 mm (main images) and 100 μm (enlargements). (B) Hydroxyproline contents in lung tissues from PN-1^−/− and PN-1^+/+ mice were measured 3, 6, and 9 days after bleomycin treatment vs saline treatment (sham). Data (mean ± SEM; n = 4-5 per group) were analyzed by 2-tailed Mann-Whitney U test. (C) Total TGFβ levels were measured by enzyme-linked immunosorbent assay in BALFs collected 9 days after saline (sham) or BLM treatment. Data (mean ± SEM; n = 10-13 per group) were analyzed by Kruskall-Wallis test with Dunn’s multiple comparison test. *P < .05, **P < .01 vs sham.

Figure 4.

Accentuated overexpression of matrix extracellular proteins in lungs of bleomycin-injured PN-1^−/−mice. Lungs from PN-1^−/− mice and their PN-1^+/+ littermates were harvested at the indicated time points after bleomycin-induced lung injury (BLM) instillation for the following analyses. Quantitative reverse transcription PCR analysis of collagen3α1 (col3α1) (A), col1α2 (B), and fibronectin (C) mRNA expressions in lung tissues. GAPDH was used as an internal control (n = 4 per group). (D) Western blot analysis of type 1 collagen (col 1) and fibronectin. GAPDH was used as a loading control. Densitometric analysis of the corresponding western blot analyses is expressed for fibronectin (E) and col 1 (F) (n = 6-10 per group). For panels A-C and E-F, data represent mean ± SEM and were analyzed by 2-tailed Mann-Whitney U test. *P < .05, **P < .01 vs respective sham.

Figure 5.

Protective effect of PN-1 from BM cells in bleomycin-injured chimeric mice. PN-1^−/− mice and their PN-1^+/+ littermates were irradiated and underwent transplantation with BM from appropriated mice and allowed to recover for 5 weeks before bleomycin-induced lung injury. PN-1^+/+ BM → PN-1^+/+: PN-1^+/+ mice receiving PN-1^+/+ BM transplants (n = 10). PN-1^−/− BM → PN-1^+/+: PN-1^+/+ mice receiving PN-1^−/− BM transplants (n = 8). PN-1^+/+ BM → PN-1^−/−: PN-1^−/− mice receiving PN-1^+/+ BM transplants (n = 7). PN-1^−/− BM → PN-1^−/−: PN-1^−/− mice receiving PN-1^−/− BM transplants (n = 5). (A) Percentages of surviving mice undergoing transplantation were plotted over a 14-day period after bleomycin treatment. Log-rank test was used to compare the difference between similar recipient mice. P = .03 for PN-1^+/+ BM → PN-1^−/− vs PN-1^−/− BM → PN-1^−/−, and P = .002 for PN-1^+/+ BM → PN-1^+/+ vs PN-1^−/− BM → PN-1^+/+. (B) Masson’s trichrome and Sirius red stainings of lung withdrawn the day of euthanasia from PN-1^+/+ and PN-1^−/− chimeric mice. Representative images are shown. Scale bars, 100 µm.

Figure 6.

Protective effect of thrombin inhibition by argatroban in bleomycin-injured PN-1^−/−mice. PN-1^−/− mice were subjected to bleomycin-induced lung injury (BLM; 2 mg/kg) and treated daily with argatroban (9 mg/kg intraperitoneally) . (A) Thrombin activity was measured by a fluorometric method. Data (mean ± SEM; n = 9-11 per group) were analyzed by Kruskall-Wallis test with Dunn’s multiple comparison test. (B) Percentages of surviving PN-1^−/− mice were plotted over a 14-day period. Log-rank test was used to compare the difference between PN-1^−/− mice with BLM and PN-1^−/− mice with BLM plus argatroban (PN-1^−/− BLM: n = 10; PN-1^−/− BLM + argatroban: n = 15; P = .02). The number of platelets (C) and WBCs (D) in BALFs were counted in the hemocytometer. Data (mean ± SEM; n = 6-9 per group) were analyzed by 1-way analysis of variance with Tukey’s multiple comparison test. *P < .05, ***P < .001, ****P < .0001.

Figure 7.

Protective effect of PAR4 expressed on hematopoietic cells in bleomycin-injured lungs. WT mice were irradiated and underwent transplantation with PN-1^−/−/Par4^+/+ or PN-1^+/+/Par4^−/− or PN-1^+/+/Par4^+/+ or PN-1^−/−/Par4^−/− BM and allowed to recover for 5 weeks before bleomycin-induced lung injury. PN-1^−/−/Par4^+/+ BM → WT (n = 10). PN-1^+/+/Par4^−/− BM → WT (n = 12). PN-1^+/+/Par4^+/+ BM → WT (n = 8). PN-1^−/−/Par4^−/− BM → WT (n = 12). (A) Percentages of surviving mice undergoing transplantation were plotted over a 14-day period after bleomycin treatment. Log-rank test was used to compare the difference between recipient mice: P = .01 for PN-1^−/−/Par4^+/+ BM → WT vs PN-1^+/+/Par4^+/+ BM → WT. (B) Masson’s trichrome and Sirius red stainings of lung withdrawn the day of euthanasia from WT chimeric mice. Representative images are shown. Scale bars, 500 µm.