Volume 140, Issue 5, Pages 1642-1652 (May 2011) Tissue Transglutaminase Does Not Affect Fibrotic Matrix Stability or Regression of Liver Fibrosis in Mice  Yury Popov, Deanna Y. Sverdlov, Anisha K. Sharma, K. Ramakrishnan Bhaskar, Shaoyong Li, Tobias L. Freitag, James Lee, Walburga Dieterich, Gerry Melino, Detlef Schuppan  Gastroenterology  Volume 140, Issue 5, Pages 1642-1652 (May 2011) DOI: 10.1053/j.gastro.2011.01.040 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Transglutaminase (TG) 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 (w) 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, ×50). (C) Time-course of TG2 messenger RNA (mRNA) expression, which (D) strongly correlates with procollagen α1(I) (COL1A1; procollagen type 1) mRNA (Pearson's test). Hepatic transcript levels were determined by quantitative reverse-transcription polymerase chain reaction and are expressed as arbitrary units (fold increase vs controls) after normalization to β2MG mRNA. (E) Total TG activity in liver homogenates. All data are mean ± standard error of mean (n = 6–8 for each bar). *P < .05 as compared to vehicle-treated controls (Ctrl) (analysis of variance). Gastroenterology 2011 140, 1642-1652DOI: (10.1053/j.gastro.2011.01.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Analysis of collagen solubility indicates increased cross-linking of the fibrotic ECM during progression of liver fibrosis. Liver collagens were solubilized from 1 g liver tissue via sequential overnight extractions with neutral salt, acetic acid, and pepsin, (A) and collagen content in each fraction was quantified biochemically via hydroxyproline determination, as described in detail in Materials and Methods. (B) Distribution of collagen fractions during fibrosis progression (0 = healthy liver; 1, 3, 6, 12 = fibrotic livers 1, 3, 6, 12 weeks after CCL4 treatment, respectively). Data represent mean ± standard error of mean from 3 samples per bar (each pooled from 2–3 individual livers for extractions and analysis). ***P > .0001 analyzed using analysis of variance with Dunnett's post-test comparing to healthy controls. Gastroenterology 2011 140, 1642-1652DOI: (10.1053/j.gastro.2011.01.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Lack of spontaneous fibrosis regression after short-term recovery in wild-type and tissue transglutaminase–deficient (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 wild-type 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 6 weeks (w) of CCL4 (B) or TAA (E) treatment and after 4 weeks of spontaneous recovery (original magnification, ×50). (C) Normalization of profibrogenic gene expression after 4 weeks of recovery. Hepatic transcript levels of procollagen type 1 (procollagen α1(I) [COL1A1]), transforming growth factor–β1 (TGFβ1) and tissue inhibitor of metalloproteinase–1 (TIMP-1) were determined by quantitative reverse-transcription polymerase chain reaction and expressed in arbitrary units (fold increase vs controls) after normalization to β2MG messenger RNA. All data are mean ± standard error of mean (n = 6–8 for each bar). *P < .05 vs vehicle controls (Ctrl) of the respective genotype. #P < .05 vs fibrotic controls (analysis of variance). Gastroenterology 2011 140, 1642-1652DOI: (10.1053/j.gastro.2011.01.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Lack of significant collagen removal after long-term recovery up to 36 weeks in WT and tissue transglutaminase–deficient (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 WT (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 mean ± standard error of mean (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 < .05 as compared to vehicle controls (Ctrl) of the respective genotype. #P < .05 vs fibrotic controls (analysis of variance). Gastroenterology 2011 140, 1642-1652DOI: (10.1053/j.gastro.2011.01.040) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Genetic deletion of TG2 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 tissue transglutaminase–deficient (TG2−/−) mice (open bars) and WT controls (closed bars) by serial extraction of liver tissue and quantification of extracted collagen in each fraction as described in Materials and Methods. 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 carbon tetrachloride [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 were found in the salt- and acid-soluble fractions, which amounted to <5% of total collagen combined (not shown). Data represent mean ± standard error of mean from 3 samples per bar (each pooled from 2–3 individual livers for extractions and analysis). Gastroenterology 2011 140, 1642-1652DOI: (10.1053/j.gastro.2011.01.040) Copyright © 2011 AGA Institute Terms and Conditions