Urokinase-type plasminogen activator supports liver repair independent of its cellular receptor

Abstract

Background: The urokinase-type (uPA) and tissue-type (tPA) plasminogen activators regulate liver matrix remodelling through the conversion of plasminogen (Plg) to the active protease plasmin. Based on the efficient activation of plasminogen when uPA is bound to its receptor (uPAR) and on the role of uPA in plasmin-mediated liver repair, we hypothesized that uPA requires uPAR for efficient liver repair.

Methods: To test this hypothesis, we administered one dose of carbon tetrachloride (CCl4) to mice with single or combined deficiencies of uPA, uPAR and tPA, and examined hepatic morphology, cellular proliferation, fibrin clearance, and hepatic proteolysis 2-14 days later.

Results: Absence of uPAR alone or the combined absence of uPAR and tPA had no impact on the resolution of centrilobular injury, but the loss of receptor-free uPA significantly impaired the clearance of necrotic hepatocytes up to 14 days after CCl4. In response to the injury, hepatocyte proliferation was normal in mice of all genotypes, except for uPAR-deficient (uPAR degrees) mice, which had a reproducible but mild decrease by 33% at day 2, with an appropriate restoration of liver mass by 7 days similar to experimental controls. Immunostaining and zymographic analysis demonstrated that uPA alone promoted fibrin clearance from centrilobular regions and efficiently activated plasminogen.

Conclusion: uPA activates plasminogen and promotes liver matrix proteolysis during repair via a process that neither requires its receptor uPAR nor requires a contribution from its functional counterpart tPA.

Figure 1

Levels of serum alanine aminotransferase (ALT) after CCl ₄ . Serum levels of ALT peak 2 days following CCl₄ administration in mice of all genotypes. Thereafter, the values return to basal levels within 7 to 14 days in a similar manner in all experimental groups (n = 3–8 at each time point). * P = 0.023 when compared experimental controls.

Figure 2

Impaired resolution of centrilobular injury in mice lacking receptor-free uPA. Histological liver sections 2 days after CCl₄ show similar features of centrilobular necrosis (arrows) in mice of all genotypes. While livers from wild type (WT), uPAR°, and uPAR°/tPA° show resolution of the centrilobular injury by day 7, uPA° livers continue to display the injury at 7 and 14 days after CCl₄ despite the presence of uPAR (Magnification 200×)

Figure 3

Transient decrease in proliferation of hepatocytes in mice lacking uPAR. Quantification of BrdU-labeled hepatocytes shows a rise in BrdU uptake in all experimental groups 2 days after CCl₄, with a decrease of ~33% in uPAR° livers. The percent of BrdU-labeled hepatocytes return to near baseline levels in all groups. WT = wild type. (*ANOVA P < 0.004; ^@t-test P < 0.004; N = 3–8 at each time point).

Figure 4

Normal activation of cell-associated hepatocyte growth factor in uPAR° livers during liver repair. Western analysis of hepatocyte growth factor (HGF) partially purified from the soluble fraction of liver extracts using an antibody that recognizes the single chain and heavy chain (to demonstrate activation) of HGF shows similar levels of activation in WT and uPAR° livers at baseline (day 0) or at 2 days after CCl₄ administration. scHGF = single chain HGF; aHGF = activated HGF, rHGF = recombinant HGF.

Figure 5

Maintenance of liver mass following CCl ₄ . Analysis of the liver mass (expressed as the wet liver weight as a percentage of the body weight) shows a mild increase in all genotypes 2 days after CCl₄ administration, followed by a trend toward baseline levels by 14 days, except for livers of uPA° mice which continue to display a mass that is similar to day 2 (and higher than before CCl₄, *P < 0.03). WT = wild type.

Figure 6

Receptor-free uPA is required for fibrin clearance. Detection of fibrin deposition by immunohistochemistry (red stain, arrows) shows uniform staining in centrilobular area in livers of mice of all genotypes 2 days after CCl₄. The clearance of fibrin coincides with the resolution of the centrilobular injury by day 14 in mice of each genotype, except for uPA° mice. (Magnification 200×)

Figure 7

Hepatic uPA activation occurs in the absence of uPAR. Activation of uPA in the liver was observed in mice of all genotypes after CCl₄, except in uPA° mice. The upper band (arrow) depicts tPA while the lower band (arrow head) depicts uPA.