ADAMTS13 Proteolysis, Hepatic Stellate Cell Activation and Liver Fibrosis in a Rat Model.
Authors: Niiya Masami , Uemura Masayuki , Wu X. , Pollak Eleanor , Dockal Michael , Scheiflinger Friedrich , Wells Rebecca , Zheng X. Long 
Published: April 2020

Abstract
ADAMTS13, the 13th member of the ADAMTS (A Disintegrin And Metalloprotease with Thrombo Spondin type 1 repeat) family, cleaves an adhesion molecule, von Willebrand factor. ADAMTS13 biosynthesis occurs primarily in liver, particularly in cells expressing alpha-smooth muscle actin, likely myofibroblasts. Hepatic stellate cells (HSCs) are one of the major cell types transdifferentiating to myofibroblasts during liver fibrosis. To explore the role of ADAMTS13 proteolysis during HSC activation and liver fibrosis, we examined: 1) the expression of Adamts13 mRNA, protein and activity in rat primary HSCs during in vitro activation; and 2) the expression of Adamts13 mRNA and activity in rat livers following induction of liver fibrosis by administration of carbon tetrachloride. Primary HSCs were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. At day 1 after isolation, the rat primary HSCs appeared small and round with abundant granules containing vitamin A droplets, traditionally defined quiescent. At day 4 and day 7 after isolation, however, cells became polymorphic (elongated or dendrictic in shape) and expressed alpha-smooth muscle actin, consistent with activation. Immunocytochemical staining with rabbit anti-ADAMTS13 showed that ADAMTS13 antigen was dramatically increased in activated rat primary HSCs compared to quiescent cells. This result was consistent with that determined by Western blotting on which a 190-kDa band was only detected in the rat HSC lysate at day 7, but not at day 1 and day 4 after isolation. However, by a more sensitive assay with FRETS-VWF73, we showed that the ADAMTS13 activity in the cell lysates of rat primary HSCs at day 4 and day 7 increased by approximately 20- and 200-fold, respectively, compared to that in rat primary HSCs at day 1 after isolation. The proteolytic activity in the cell lysates was completely inhibited by EDTA (10 mM) and rabbit anti-ADAMTS13 IgG (40 g/ml), suggesting the specificity of substrate cleavage by ADAMTS13. In contrast, the Adamts13 mRNA in the rat primary HSCs at day 4 and day 7 increased only by about 1.3-fold at day 4 and 1.5-fold, respectively, compared to that at day 1 after isolation. Furthermore, ADAMTS13 activity in the liver tissue lysates obtained from rats at day 10 (mid liver fibrosis seen) and at day 70 (severe liver fibrosis seen) after administration of carbon tetrachloride also increased by about 5-fold and 115-fold, respectively, compared to that in the normal control. As it was found in the primary rat HSCs, the Adamts13 mRNA in the liver tissues with mild fibrosis and severe fibrosis only increased by about 2.5-fold and 3.0-fold, respectively, compared to that in the normal liver tissues. Such a dramatic discrepancy between the increment of Adamts13 mRNA and proteolytic activity in the rat primary HSCs upon activation and in the liver tissues after administration of carbon tetrachloride suggests the possibility of posttranslational modifications of ADAMTS13 and/or activation of ADAMTS13 protease. The significantly enhanced ADAMTS13 proteolysis in rat primary HSCs upon activation and during liver injury may be important in remodeling extracellular matrix components to accelerate the resolution of liver fibrosis. Our data also suggest that HSC may be an excellent model to study the biosynthesis and activation of ADAMTS13 metalloprotease.