162 Progressive Matrix Crosslinking Limits Reversibility of Liver Fibrosis and is Independent of Tissue Transglutaminase

Abstract

Background: Targeting extracellular matrix stability is an attractive antifibrotic strategy. Prior studies have suggested a role for tissue transglutaminase-mediated collagen crosslinking in matrix stabilization, but the extent and mechanisms remain poorly understood. We therefore used a quantitative biochemical approach to characterize dynamic changes in collagen crosslinking during progression and reversal of CCL4-induced liver fibrosis in wildtype mice and in mice deficient in transglutaminase 2 (TG2), the predominant transglutaminase isoform in the liver. Materials and Methods: Liver fibrosis was induced in TG2-/mice and wildtype littermates (C57BL6) by repeated gavage with carbon tetrachloride (CCL4) 3x per week for 1, 3, 6 and 12 weeks. Fibrosis reversal was monitored for up to 36 weeks after CCL4 withdrawal. Collagen crosslinking was measured by sequential extraction using neutral salt, acetic acid and pepsin. Collagen was quantified biochemically via hydroxyproline in the livers, in the extracts and in residual liver tissue. This was correlated to histological evolution and transcript levels of fibrosis related genes. Results: In normal liver, neutral salt, acetic acid and pepsin solubilized 3.2, 1.2% and 91% of total collagen, respectively, while 4.5% remained insoluble, representing a highly cross-linked collagenous matrix. CCL4 administration for 12w led to development of cirrhosis and a 6-fold increase in total liver collagen. Collagen solubility during fibrosis progression revealed progressive cross-linking, with an increase of the insoluble collagen fraction to 27% (6-fold) at week 12 at the expense of the pepsin-soluble fraction. The saltand acid-soluble collagen fractions, representing freshly synthesized collagen, remained constant at 5% of total collagen. The extent of matrix deposition and stabilization remained constant up to 36 weeks after discontinuation of CCL4. Furthermore, no difference in collagen solubility and no fibrosis reversal or change in matrix-related transcripts were found in TG2-/vs. wildtype mice during all time points of progression or follow-up. Conclusions: 1) Quantitative measurement of fibrotic matrix stability revealed a significant increase of collagen crosslinking, which parallels fibrosis progression and limits its reversal. 2) Matrix stabilization in fibrotic liver is independent of transglutaminase-mediated cross-linking since it was unaffected in TG2-/mice 3) This TG2independent collagen cross-linking is an important target for future antifibrotic therapies.