Plasminogen activator inhibitor-1 limits liver injury and facilitates regeneration after acetaminophen overdose

Abstract

Deficiency in plasminogen activator inhibitor-1 (PAI-1) gene expression is known to promote growth factor activation and regeneration in a number of hepatotoxicity models. To evaluate if PAI-1 has similar effects in acetaminophen (APAP) hepatotoxicity, wild-type (WT) and PAI-1 gene knockout mice (PAI-KO) were treated with 200 mg/kg APAP and liver injury and its repair were assessed. In WT animals, plasma alanine aminotransferase (ALT) activities increased during the first 12 h and then returned to baseline within 48 h. The area of necrosis increased in parallel to the ALT values, peaked between 12 and 24 h and was completely resolved by 96 h. The regenerative response of cells outside the necrotic area, as indicated by proliferating cell nuclear antigen protein and cyclin D(1) gene expression, was observed within 24 h, peaked at 48 h and then declined but remained elevated until 96 h. Liver injury in response to APAP was similar in PAI-KO as in WT animals during the first 12 h. However, plasma ALT values and the area of necrosis further increased during the following 12 h with development of massive intrahepatic hemorrhage. Approximately, 50% of the PAI-KO animals did not survive. Although liver injury of the surviving animals was repaired, the regeneration process was delayed until 48 h. A potential reason for this delay may have been due to the more severe injury and/or the increased expression of the cell cycle inhibitor p21. Our data indicate that PAI activation limits liver injury and mortality during APAP hepatotoxicity by preventing excessive hemorrhage and thereby facilitating tissue repair.

FIG. 1.

Time course of PAI-1 mRNA expression in WT and PAI-KO mice before and after 200 mg/kg APAP treatment. Data are expressed as the PAI-to-actin mRNA ratio; the value of WT control animals was set as 100%. The mean ± SE of n = 4–6 animals per time point is shown. *p < 0.05 (compared with PAI-KO mice).

FIG. 2.

Hepatic glutathione levels were determined in animals 20 min after treatment with either the vehicle saline (S) or 200 mg/kg APAP. WT and PAI-KO mice were compared. Data represent the mean ± SE of n = 4 animals per group. *p < 0.05 (compared with the respective vehicle group).

FIG. 3.

Plasma ALT activities (A) and the area of necrosis (B) were measured as indicators for APAP-induced liver injury in WT and in PAI-KO animals. Animals received 200 mg/kg APAP at t = 0. Data represent means ± SE of n = 4–6 animals per group. *p < 0.05 (compared with WT mice).

FIG. 4.

Representative liver sections stained with H&E from APAP-treated animals (200 mg/kg). Sections were taken from the 24 h (A, B) and the 48 h (C, D) groups of WT (A, C) and PAI-KO mice (B, D). Sections show extensive necrosis around the centrilobular areas (*) of WT and KO mice and severe hemorrhage only in PAI-KO mice. Arrows indicate periportal areas (×100).

FIG. 5.

DNA fragmentation was assessed by the TUNEL assay in WT (A, C) and in PAI-KO mice (B, D) at 12 h (A, B) and 24 h (C, D) after APAP (200 mg/kg) treatment. The panels show TUNEL-positive cells around the centrilobular areas (×50).

FIG. 6.

Western blot analysis of PCNA expression in livers of controls or animals treated with 200 mg/kg APAP for 24 h (A) or 48 h (B). Livers were obtained from WT animals and PAI-KO mice. One to three representative samples are shown from each group. (C) Densitometric analysis of PCNA western blots from controls and APAP-treated WT and PAI-KO mice. Data of control animals were set as 100%. All data represent means ± SE of n = 4–6 animals per time point. *p < 0.05 (compared with controls), ^#p < 0.05 (compared with WT).

FIG. 7.

Representative liver sections obtained from APAP-treated animals (200 mg/kg) were stained for PCNA. Sections were taken from the 24 h (A, B) and the 48 h (C, D) groups of WT (A, C) and PAI-KO mice (B, D). Sections show extensive PCNA staining (dark brown nuclei) outside the areas of centrilobular necrosis (*) of WT (24, 48 h) and KO mice (48 h only). #Indicates periportal areas (×100).

FIG. 8.

Time course of mRNA expression of cyclin D₁ (A) and p21 (B) before and after treatment with 200 mg/kg APAP in WT animals and PAI-KO mice. Cyclin D₁ and p21 mRNA levels were compared with the housekeeping gene β-actin and the gene-to-actin ratio is reported as percent of control animals (t =0 h). All data represent means ± SE of n = 4–6 animals per time point. *p < 0.05 (compared with controls), ^#p < 0.05 (compared with WT).