Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice

Abstract

Background & aims: The ubiquitous cross-linking enzyme tissue transglutaminase (TG2) has been implicated in irreversible collagen stabilization in liver fibrosis, although functional evidence is lacking. We studied the contribution of TG2 to hepatic fibrotic matrix stability, as well as liver fibrosis progression and regression in TG2-deficient mice.

Methods: Advanced liver fibrosis was induced by carbon tetrachloride or thioacetamide in TG2(-/-) mice and their wild-type littermates to study fibrosis progression and its spontaneous regression for up to 36 weeks. Pattern and extent of fibrosis were analyzed by histology and hepatic hydroxyproline quantification. Dynamic changes in hepatic matrix cross-linking were assessed by stepwise collagen extraction. Expression of 7 TGs and fibrosis-related genes was determined by quantitative reverse-transcription polymerase chain reaction.

Results: Transglutaminase activity was increased in fibrosis, and the level of TG2 messenger RNA correlated with the expression of fibrosis-related genes. Biochemical analysis revealed progressive collagen stabilization, with an up to 6-fold increase in the highly cross-linked, pepsin-insoluble fraction (26%). In TG2(-/-) mice, hepatic TG activity was significantly decreased, but chronic administration of carbon tetrachloride or thioacetamide led to a comparable extent and pattern of liver fibrosis, as in wild-type mice. In TG2(-/-) mice, the composition of hepatic collagen fractions and levels of fibrosis-related transcripts were unchanged, and fibrosis reversal was not facilitated.

Conclusions: TG2 and TG activity are up-regulated during hepatic fibrosis progression, but do not contribute to fibrogenesis or stabilization of the collagen matrix. TG2 deletion does not promote regression of liver fibrosis. TG2-independent collagen cross-linking is a remarkable feature of progressing hepatic fibrosis and represents an important therapeutic target for liver fibrosis.

Figure 1. Tranglutaminase expression and activity is elevated in progressive CCL4-induced liver fibrosis and correlates with fibrogenesis activity

A) Total hepatic collagen content progressively increases at 1, 3, 6 and 12 weeks after CCL4 treatment in C57Bl/6 mice as determined biochemically via hepatic hydroxyproline. B) Low magnification images of connective tissue stain (Sirius Red) of representative sections from livers at 1, 3, 6 and 12 weeks after CCL4 treatment (original magnification, x50). C) Time-course of TG2 mRNA expression, which D) strongly correlates with COL1A1 (procollagen type 1) mRNA (Pearson’s test).Hepatic transcript levels were determined by quantitative RT-PCR, and are expressed as arbitrary units (fold increase vs. controls) after normalization to β2MG mRNA. (E) Total transglutaminase activity in liver homogenates. All data are means±SEM(n=6-8 for each bar) *, p<0.05 as compared to vehicle treated controls (Ctrl) (ANOVA).

Figure 2. Analysis of collagen solubility indicates increased cross-linking of the fibrotic ECM during progression of liver fibrosis

A) Liver collagens were solubilized from 1g of liver tissue via sequential overnight extractions with neutral salt, acetic acid and pepsin, and collagen content in each fraction was quantified biochemically via hydroxyproline determination, as described in detail in M&M(0=healthy liver; 1,3,6,12=fibrotic livers 1, 3, 6, 12 weeks after CCL4 treatment, respectively). Data represent means±SEM from 3 samples per bar (each pooled from 2-3 individual livers for extractions and analysis). ***, p values >0.0001 analyzed using ANOVA with Dunnet’s post-test comparing to healthy controls.

Figure 3. Lack of spontaneous fibrosis regression after short-term recovery in wildtype and TG2−/− mice with advanced liver fibrosis induced with CCL4 or TAA

Total hepatic collagen content at the peak of fibrosis induced by CCL4 (A) or TAA (D) for 6 weeks, and after 4 weeks of recovery in TG2−/− (open bars) mice and their wildtype littermates (closed bars), as determined biochemically via hepatic hydroxyproline. B) Low magnification images of connective tissue stain (Sirius Red) of representative sections from livers at 6w of CCL4 (B) or TAA (E) treatment and after 4 weeks of spontaneous recovery (original magnification, ×50). C) Normalization of profibrogenic gene expression after 4w of recovery. Hepatic transcript levels of procollagen type 1(COL1A1), TGFβ1 and TIMP-1 were determined by quantitative RT-PCR, and expressed in arbitrary units (fold increase vs. controls) after normalization to β2MG mRNA. All data are means±SEM(n=6-8 for each bar) *, p<0.05 vs. The vehicle controls (Ctrl) of the respective genotype. #, p<0.05 vs. the fibrotic controls (ANOVA).

Figure 4. Lack of significant collagen removal after long-term recovery up to 36 weeks in wildtype and TG2−/− mice with advanced CCL4-induced liver fibrosis

Relative (A) and total (B) hepatic collagen content at the peak of fibrosis induced with CCL4 for 6 weeks, and after 8, 12, 24 and 36 weeks of recovery in wildtype (closed bars) and TG2−/− (open bars) C57Bl/6 mice, as determined biochemically via hepatic hydroxyproline. C) Compensatory increase in liver volume during recovery coincides with a decrease in relative, but not total, hepatic collagen content as measured by liver weight at sacrifice. All data are means±SEM(n=6-10 for each bar). D) High magnification images of collagen (Sirius Red) of representative sections from livers at 6 weeks of CCL4 treatment and after 36 weeks of spontaneous recovery (original magnification, ×200). E) Hepatocytes invading fibrotic septa are a characteristic feature observed during long-term recovery (WT mice after 8 weeks of recovery shown, original magnification as indicated). F) Morphometric analysis of scar tissue remodeling during recovery demonstrates widening of septa and splitting of condensed collagen bundles into thinner fibrils. Measurements were performed at ×200 magnification, using an ocular net micrometer. Thickness of septa and number of fibrils was assessed at outer boundaries of the middle third of at least 10 randomly selected complete fibrotic septa in specimens from a right and a left liver lobe and from 4 individual mice/group at peak fibrosis and at 4 weeks of resolution). *, p<0.05 as compared to vehicle controls (Ctrl) of the respective genotype. #, p<0.05 vs. the fibrotic controls (ANOVA).

Figure 5. Genetic deletion of tTG does not affect solubility of collagen in mice without fibrosis, and mice with advanced fibrosis or after long-term recovery

Fibrotic ECM solubility was determined in TG2−/− mice (open bars) and wildtype controls (closed bars) by serial extraction of liver tissue and quantification of extracted collagen in each fraction as described in M&M. Only the quantitatively relevant pepsin-soluble and insoluble fractions are shown. Ctrl, vehicle-treated, nonfibrotic controls. “Fibrosis”, fibrotic mice at the peak of fibrosis (6 weeks of CCL4 treatment). “Recovery”, CCL4 treatment was stopped, and fibrotic mice were allowed to recover for an additional 36 weeks. No differences between genotypes or treatments was found in the salt- and acid-soluble fractions, which amounted to less than 5% of total collagen combined (not shown). Data represent means±SEM from 3 samples per bar (each pooled from 2-3 individual livers for extractions and analysis).