Elsevier

Journal of Hepatology

Volume 66, Issue 4, April 2017, Pages 787-797
Journal of Hepatology

Research Article
Fibrin(ogen) drives repair after acetaminophen-induced liver injury via leukocyte αMβ2 integrin-dependent upregulation of Mmp12

https://doi.org/10.1016/j.jhep.2016.12.004Get rights and content

Background & Aims

Acetaminophen (APAP)-induced liver injury is coupled with activation of the blood coagulation cascade and fibrin(ogen) accumulation within APAP-injured livers of experimental mice. We sought to define the role of fibrin(ogen) deposition in APAP-induced liver injury and repair.

Methods

Wild-type, fibrinogen-deficient mice, mutant mice with fibrin(ogen) incapable of binding leukocyte αMβ2 integrin (Fibγ390-396A mice) and matrix metalloproteinase 12 (Mmp12)-deficient mice were fasted, injected with 300 mg/kg APAP i.p. and evaluated at a range of time-points. Plasma and liver tissue were analyzed. Rescue of Fibγ390-396A mice was carried out with exogenous Mmp12. To examine the effect of the allosteric leukocyte integrin αMβ2 activator leukadherin-1 (LA-1), APAP-treated mice were injected with LA-1.

Results

In wild-type mice, APAP overdose increased intrahepatic levels of high molecular weight cross-linked fibrin(ogen). Anticoagulation reduced early APAP hepatotoxicity (6 h), but increased hepatic injury at 24 h, implying a protective role for coagulation at the onset of repair. Complete fibrin(ogen) deficiency delayed liver repair after APAP overdose, evidenced by a reduction of proliferating hepatocytes (24 h) and unresolved hepatocellular necrosis (48 and 72 h). Fibγ390-396A mice had decreased hepatocyte proliferation and increased multiple indices of liver injury, suggesting a mechanism related to fibrin(ogen)-leukocyte interaction. Induction of Mmp12, was dramatically reduced in APAP-treated Fibγ390-396A mice. Mice lacking Mmp12 displayed exacerbated APAP-induced liver injury, resembling Fibγ390-396A mice. In contrast, administration of LA-1 enhanced hepatic Mmp12 mRNA and reduced necrosis in APAP-treated mice. Further, administration of recombinant Mmp12 protein to APAP-treated Fibγ390-396A mice restored hepatocyte proliferation.

Conclusions

These studies highlight a novel pathway of liver repair after APAP overdose, mediated by fibrin(ogen)-αMβ2 integrin engagement, and demonstrate a protective role of Mmp12 expression after APAP overdose.

Lay summary

Acetaminophen overdose leads to activation of coagulation cascade and deposition of high molecular weight cross-linked fibrin(ogen) species in the liver. Fibrin(ogen) is required for stimulating liver repair after acetaminophen overdose. The mechanism whereby fibrin(ogen) drives liver repair after acetaminophen overdose requires engagement of leukocyte αMβ2 integrin and subsequent induction of matrix metalloproteinase 12.

Introduction

Acute liver injury caused by acetaminophen (APAP) overdose in experimental animals is associated with activation of the coagulation cascade. Strong evidence also connects APAP-induced liver injury in humans to changes in the hemostatic system including thrombocytopenia, elevated plasma levels of thrombin-antithrombin (TAT), increased levels of procoagulant microvesicles, and a reduction in plasma fibrinogen concentration [1], [2], [3]. Prolongation of the prothrombin time-international normalized ratio (PT-INR) is linked with increased morbidity in patients with acute liver failure, whereas preservation of plasma fibrinogen is coupled to improved outcome [2], [4], [5], [6]. Many of these changes are perceived as a direct consequence of acute liver damage, potentially reflecting failed synthesis of coagulation factors, such as fibrinogen, by the injured liver. However, there is accumulating experimental evidence that coagulation factors themselves are powerful modifiers of acute APAP hepatotoxicity.

A hepatotoxic dose of APAP administered experimentally to mice recapitulates many of the perceived changes in coagulation observed in APAP-poisoned patients. Although early experimental APAP hepatotoxicity is independent of fibrin(ogen), we and others have observed profound deposition of fibrin(ogen) within areas of hepatocellular necrosis [7], [8]. The fibrin(ogen) gamma chain can engage the leukocyte integrin αMβ2 (Mac-1, CD11b/CD18) and through this mechanism, fibrin(ogen) can define the activity of inflammatory cells such as macrophages [9], [10]. Notably, macrophages infiltrate the liver after APAP overdose in humans and mice [11], [12]. Further, strong evidence suggests that macrophages play a central role in liver repair after APAP overdose [13], [14]. However, to date the precise signals required to trigger leukocyte-driven liver repair after APAP overdose have not been identified, and the potential for fibrin(ogen) to act in this capacity has largely been overlooked.

We tested the hypothesis that fibrin(ogen)-integrin αMβ2 interaction contributes to liver repair after APAP overdose. To test this hypothesis, we utilized the thrombin inhibitor dabigatran etexilate, mice completely lacking fibrin(ogen), mice expressing a mutant fibrin(ogen) incapable of binding leukocyte integrin αMβ2, and an allosteric agonist of αMβ2. Moreover, we investigated matrix metalloproteinase 12 (Mmp12) induction as a fibrin(ogen)-integrin αMβ2 driven event central to liver repair after APAP overdose.

Section snippets

Mice

Fibrinogen Aα-deficient (Fib−/−) and fibrinogen heterozygous control mice (Fib+/−) have been described previously [15]. Bleeding is observed in pregnant Fib−/− female mice. Therefore, Fib−/− mice were maintained by breeding male Fib−/− mice with female Fib+/− mice, a strategy employed by numerous previous studies to compare fibrin(ogen)-sufficient mice with mice lacking circulating fibrin(ogen) [8], [16], [17], [18]. Mice expressing a mutant form of fibrinogen γ390-396A (Fibγ390-396A) lacking a

Intrahepatic deposition of low molecular weight and high molecular weight urea-insoluble cross-linked fibrin(ogen) after APAP overdose

Plasma fibrinogen levels decreased over time in mice administered a hepatotoxic dose of APAP (300 mg/kg) relative to vehicle-treated animals (Fig. 1A). This was paralleled by fibrin(ogen) deposition in the livers of APAP-treated mice at 24 h, which immunohistochemistry detected primarily within areas of hepatocellular necrosis (Fig. 1B). Analysis of mouse hepatic extracts revealed that levels of thrombin-cleaved fibrinogen beta chain increased 24 h after APAP challenge, suggesting that the observed

Discussion

Several studies link coagulation cascade activation to exacerbation of early (6 h) APAP hepatotoxicity in mice, with thrombin activation of PAR-1 and PAR-4 identified as the primary mechanism [7], [8], [25]. However, despite a profound reduction in coagulation, tissue factor (TF) deficiency offered only transient protection from APAP-induced liver damage [7], and here we identified that prolonged anticoagulation with DABI ultimately reduced hepatocyte proliferation and increased liver damage.

Financial support

This work was supported by the National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [R01 DK087886 and R01 DK105099]. JEF and BFJ were supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy through grant no. DE–FG02–91ER20021 (to JEF). The content is solely the responsibility of the authors and does not necessarily represent the official

Conflict of interest

JPL has a sponsored research grant from Boehringer-Ingelheim, which is unrelated to the manuscript. Boehringer-Ingelheim did not provide material or financial support for the work described.

Authors’ contributions

Designed and conducted experiments: AKK, NJ, HC-F, AVW, JLR, BPS, JEF, BFJ, MJF, JPL. Analyzed data: AKK, JEF, BFJ, MJF, JPL. Drafted initial sections of the manuscript: AKK, MJF, JPL. Approved final version of the manuscript: AKK, NJ, HC-F, AVW, JLR, BPS, JEF, BFJ, MJF, JPL.

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