Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis

Abstract

Fibrotic lung disease, most notably idiopathic pulmonary fibrosis (IPF), is thought to result from aberrant wound-healing responses to repetitive lung injury. Increased vascular permeability is a cardinal response to tissue injury, but whether it is mechanistically linked to lung fibrosis is unknown. We previously described a model in which exaggeration of vascular leak after lung injury shifts the outcome of wound-healing responses from normal repair to pathological fibrosis. Here we report that the fibrosis produced in this model is highly dependent on thrombin activity and its downstream signaling pathways. Direct thrombin inhibition with dabigatran significantly inhibited protease-activated receptor-1 (PAR1) activation, integrin αvβ6 induction, TGF-β activation, and the development of pulmonary fibrosis in this vascular leak-dependent model. We used a potentially novel imaging method - ultashort echo time (UTE) lung magnetic resonance imaging (MRI) with the gadolinium-based, fibrin-specific probe EP-2104R - to directly visualize fibrin accumulation in injured mouse lungs, and to correlate the antifibrotic effects of dabigatran with attenuation of fibrin deposition. We found that inhibition of the profibrotic effects of thrombin can be uncoupled from inhibition of hemostasis, as therapeutic anticoagulation with warfarin failed to downregulate the PAR1/αvβ6/TGF-β axis or significantly protect against fibrosis. These findings have direct and important clinical implications, given recent findings that warfarin treatment is not beneficial in IPF, and the clinical availability of direct thrombin inhibitors that our data suggest could benefit these patients.

Keywords: Inflammation; Pulmonology.

Figure 1. Thrombin inhibition attenuates fibrosis in a vascular leak–dependent model.

(A) Plasma dilute thrombin times in mice treated with dabigatran (Dabi) or vehicle (Veh) for 1 week (representative of 2 independent experiments). (B and C) Representative images of Masson’s trichrome–stained mouse lung sections at day 14 after bleomycin + FTY720 challenge with vehicle (B) or dabigatran (C) treatment (n = 3/group). Original magnification, ×40. (D–F) Measurement of total lung hydroxyproline content (D), bronchoalveolar lavage (BAL) total protein concentration (E), and BAL total leukocytes (F) at day 7 (D7) and/or D14 in mice challenged with intratracheal (i.t.) PBS + i.p. sterile water (control) or i.t. bleomycin + i.p. FTY720 (Bleo/FTY) and treated with dabigatran or vehicle (hydroxyproline data are representative of 11 independent experiments). Individual data points are presented, along with as mean ± SEM. **P = 0.006, ***P < 0.0001 by 2-tailed t tests.

Figure 2. Thrombin inhibition attenuates lung fibrin deposition.

(A) Extravascular lung D-dimer content at day 14 in control mice and mice challenged with bleomycin + FTY720 and treated with vehicle (Veh) or dabigatran (Dab). (B–F) Data from ultrashort echo time (UTE) lung MRI with the gadolinium-based, fibrin-specific probe EP-2104R at day 14 in control mice and mice challenged with bleomycin + FTY720 and treated with vehicle or dabigatran (n = 4–5/group). (B) Representative images from EP-2104R–enhanced lung MRI. (C) Semiquantitative analysis of the imaging data from EP-2104R–enhanced UTE lung MRI. (D) Quantitative assessment of lung fibrin deposition by measurement of total lung EP-2104R content. (E) Correlation between total lung D-dimer content and total lung EP-2104R content of mice challenged with bleomycin + FTY720 and treated with dabigatran versus vehicle. (F) Correlation between total lung hydroxyproline content and total lung EP-2104R content in mice challenged with bleomycin + FTY720 and treated with dabigatran versus vehicle. Data are representative of 2 independent experiments. Individual data points are presented in graphs, along with mean ± SEM where applicable. *P < 0.05, **P < 0.005, ***P < 0.0005 by 2-tailed t tests for indicated pairwise comparisons.

Figure 3. Therapeutic anticoagulation with warfarin does not protect against fibrosis.

(A) Mouse plasma international normalized ratios (INRs) after treatment with warfarin or vehicle for 2 weeks. (B and C) Measurement of lung extravascular D-dimer content (B) and total lung hydroxyproline content (C) at day 14 in control mice and mice challenged with bleomycin + FTY720 and treated with vehicle (Veh) or warfarin (War) (hydroxyproline data are representative of 11 independent experiments). (D) Correlation between total lung hydroxyproline content and total lung D-dimer content in mice challenged with bleomycin + FTY720 and treated with warfarin versus vehicle. (E–G) Western blot analyses for cleaved (activated) PAR1 in whole-lung homogenates at day 14 in control mice and mice challenged with bleomycin + FTY720 and treated with dabigatran (Dabi) versus vehicle (E, top panel; densitometry in F) or warfarin versus vehicle (E, bottom panel; densitometry in G). Western blot bands for cleaved PAR1 and GAPDH were seen at the expected molecular weights of ~46 kDa and ~37 kDa, respectively. Western blot data are representative of at least 2 independent experiments for each analysis. Individual data points are presented in graphs, along with mean ± SEM where applicable. *P < 0.05, **P < 0.005 by 2-tailed t tests for indicated pairwise comparisons.

Figure 4. Expression of α_vβ₆ integrin in a vascular leak–dependent model of lung fibrosis.

(A–F) Representative images of immunohistochemical staining for the α_vβ₆ integrin in day 14 lung sections from control mice (A and B) and mice challenged with bleomycin + FTY720 and treated with vehicle (Veh) (C and D) or dabigatran (Dab) (E and F). n = 3/group. Scale bars: 50 μm.

Figure 5. Effects of thrombin inhibition and vitamin K antagonism on lung α_vβ₆ expression and TGF-β signaling.

(A–C) Western blot analyses for α_vβ₆ (A, top panel; densitometry in B), SMAD2 and phosphorylated SMAD2 (p-SMAD2) (A, bottom panel; densitometry in C) in whole-lung homogenates at day 14 in control mice and mice challenged with bleomycin + FTY720 and treated with dabigatran versus vehicle. (D–F) Western blot analyses for α_vβ₆ (D, top panel; densitometry in E), SMAD2 and p-SMAD2 (D, bottom panel; densitometry in F) in whole-lung homogenates at day 14 in control mice and mice challenged with bleomycin + FTY720 and treated with warfarin versus vehicle. Western blot bands for α_vβ₆, SMAD2, and p-SMAD2 were seen at the expected molecular weights of ~80 kDa, ~60 kDa, and ~60 kDa, respectively; bands for β-actin and GAPDH were seen at the expected molecular weights of ~45 kDa and ~37 kDa, respectively. Data are representative of at least 2 independent experiments for each analysis. Individual data points presented in graphs, along with mean ± SEM where applicable. *P = 0.01, **P = 0.008, ***P < 0.0005 by 2-tailed t tests for indicated pairwise comparisons.

Figure 6. Antibody blockade of α_vβ₆ protects against lung fibrosis in a vascular leak–dependent model.

(A–C) Measurement of total lung hydroxyproline content (A), bronchoalveolar lavage (BAL) total protein concentration (B), and BAL total leukocytes (C) at day 14 (D14) in mice challenged with intratracheal (i.t.) PBS + i.p. sterile water (control) or i.t. bleomycin + i.p. FTY720 (Bleo/FTY) and treated with the α_vβ₆-blocking antibody (3G9) versus isotype control antibody (1E6) at 1 mg/kg s.c. 3 times per week. Data are representative of 4 independent experiments. Individual data points are presented, along with mean ± SEM. **P = 0.006 by 2-tailed t test.

Figure 7. Schematic of the proposed mechanisms linking vascular leak, intra-alveolar thrombin, α_vβ₆, and TGF-β signaling with the development of injury-induced lung fibrosis.

As a consequence of lung injury, there is damage to the alveolar epithelium, denudement of the basement membrane, and increased vascular permeability, with extravasation of plasma constituents into the injured alveoli (orange arrows). Among these extravasated plasma contents are the clotting factors, leading to intra-alveolar activation of the coagulation cascade and the generation of active thrombin. In addition to its proteolytic cleavage of fibrinogen to generate fibrin in the airspaces, thrombin also cleaves and activates proteinase activated receptor 1 (PAR1). Based on previous work by Jenkins et al. (ref. 26) and Munger et al. (ref. 27), it has been shown that activation of PAR1 on alveolar epithelial cells consequently activates the α_vβ₆ integrin (in a manner dependent on RhoA and Rho kinase, a.k.a. ROCK), which results in the release of extracellular active TGF-β from the latency-associated peptide (LAP). In an autocrine fashion, active TGF-β can then signal through its receptors (TGF-βR; here shown on a neighboring fibroblast) to exert its profibrotic effects. Dabigatran, a direct thrombin inhibitor (DTI), or blockade of the α_vβ₆ integrin interrupts this thrombin/PAR1/α_vβ₆/TGF-β axis, thereby halting the progression from lung injury to fibrosis. Conversely, while warfarin is effective at reducing intra-alveolar fibrin accumulation (solid red line), it appears to have only weak effects on overall thrombin activity (dashed red line) in the injured airspaces, which may explain its inability to significantly attenuate the development of fibrosis after lung injury.