Liver cirrhosis as a consequence of many forms of

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1 GASTROENTEROLOGY 2011;140: 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, and DETLEF SCHUPPAN* *Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; Molecular Biology Core Facilities, Dana-Farber Cancer Institute, Boston, Massachusetts; Department of Medicine 1, University of Erlangen-Nuremberg, Erlangen, Germany; University of Tor Vergata, Rome, Italy See editorial on page 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 crosslinked, 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. Keywords: Cirrhosis; Liver Disease; Mouse Model; Fibrosis Gene Expression. Liver cirrhosis as a consequence of many forms of chronic liver diseases is associated with high morbidity and mortality. 1 Causal treatment for advanced fibrosis and cirrhosis is unsatisfactory or inefficient. Therefore, antifibrotic therapies to treat and reverse fibrosis are urgently needed. 2 Although most antifibrotic strategies are directed against hepatic stellate cell/myofibroblast proliferation and profibrogenic activation, 3 only a few smaller studies have targeted extracellular matrix (ECM) stabilization, mainly the cross-linking of collagen. Targeting the process of fibrotic ECM stabilization therapeutically would indeed be a very attractive approach to induce fibrosis regression or even reversibility, eg, after successful treatment of the underlying cause of a given chronic liver disease. In vivo stabilization of the fibrotic ECM is achieved by cross-linking of the abundant (fibrillar) collagens type I, III, and V, which can represent 50% of the fibrotic liver tissue 4 and of other ECM molecules, such as elastin and fibronectin. Stabilization of the collagenous and elastic ECM can occur via 3 distinct processes, ie, the enzymatic action of lysyl oxidase or transglutaminase (mainly tissue transglutaminase [TG2]), or driven by age and hyperglycemia via nonenzymatic glycation and lipid peroxidation. Lysyl oxidase (EC , encoded by the lysyl oxidase gene 5 ) oxidizes the side chain of peptidyl lysine, resulting in residues of -aminoadipic- -semialdehyde, which permits the covalent cross-linking of the component chains of collagen and elastin. 6 The family of TGs (EC ) consists of 8 family members (TG1 7 and factor XIII 7 ), of which TG2 is the most ubiquitously expressed and abundant enzyme. TG2 and most other family members catalyze the formation of -( -glutamyl) lysine isopeptide cross-links between a donor protein with a particular glutamine residue and a more general acceptor protein. TG2-catalyzed isopeptide bond formation has been de- Abbreviations used in this paper: CCL4, carbon tetrachloride; ECM, extracellular matrix; IP, intraperitoneal; MMP, matrix metalloproteinase; TAA, thioacetamide; TG, transglutaminase; WT, wild-type by the AGA Institute /$36.00 doi: /j.gastro

2 May 2011 TISSUE TRANSGLUTAMINASE IN LIVER FIBROSIS 1643 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 1(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 2MG 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). scribed for some ECM glutamine-donor proteins, specifically several collagens (via their telopeptide extensions), procollagen type III (via the N-propeptide), nidogen, and fibronectin. 8 TGs, especially TG2, are involved in several human diseases, 9,10 in wound healing and fibrosis, 11 and represent promising pharmacological targets. TG2 is overexpressed in human liver fibrosis and cirrhosis of various etiologies, and TG-catalyzed -( glutamyl) lysine isopeptide cross-links can be detected in hepatic scar tissue. 12 These findings are consistent with descriptive in vivo data in carbon tetrachloride (CCL4) induced liver fibrosis in rats, which suggested that the limited reversibility of advanced fibrosis after toxin withdrawal can be explained by TG2-mediated collagen cross-linking. 13 Moreover, in vitro TG2-mediated cross-linking can render collagen type I resistant to the action of collagenases. Thus, cleavage of collagen type I secreted by dermal fibroblasts by interstitial collagenase (matrix metalloproteinase 1 [MMP-1]) was reduced by 50% in the presence of recombinant TG2, 14 and cross-linking of rat tail collagen type I with TG2 increased its resistance to proteolytic degradation by MMP-1 and MMP However, although involvement of TG2 in promoting fibrosis and limiting its reversibility is plausible, the contribution of TG2 to fibrotic ECM stabilization or to reversibility of hepatic fibrosis in vivo has not been addressed in functional studies. Because TG inhibitors, including agents specific for TG2, have already been developed as treatments of celiac disease, 16 validation of TG2 as a relevant antifibrotic drug target for patients with fibrotic liver diseases is of high interest. 3 In this study, we focused on the role of TG2 in stabilization of fibrotic ECM and reversibility of liver fibrosis in vivo. First, we characterized TG2 expression and activity in relation to fibrosis progression and stage, collagen

3 1644 POPOV ET AL GASTROENTEROLOGY Vol. 140, No. 5 accumulation and ECM turnover in 2 models of murine liver fibrosis in vivo. Second, we quantitatively assessed the dynamic changes in ECM stability using stepwise collagen extractions. Finally, we studied how TG2 deficiency affects ECM stability and fibrosis reversibility by comparing TG2 / mice with their wild-type (WT) littermates. Materials and Methods Animal Experiments TG2-deficient mice were generated and characterized as described in detail previously 17 and backcrossed onto the C57Bl/6J background (Jackson Laboratories, Bar Harbor, ME) for 12 generations. Animals were housed on a 12-hour dark and light cycle and fed a standard rodent chow and tap water ad libitum. WT age- and sex-matched littermates from the TG2 / colony, and WT C57Bl/6J mice purchased from Jackson Laboratories were used as controls. No difference in fibrosis susceptibility was found between controls from our colony and from Jackson (not shown). Animal experiments had been approved by the Institutional Animal Care and Use Committee (BIDMC, protocol # ). Optimization of hepatotoxin-induced liver fibrosis models in C57Bl/6 mouse. Hepatotoxin-induced liver fibrosis was induced in 7- to 8-week-old mice by chronic injections of carbon tetrachloride (CCL4) or thioacetamide (TAA) and spontaneous recovery was monitored short term (4 weeks) and long term (8 36 weeks) after withdrawal of the hepatotoxin. First, optimal dosing regimens for fibrosis induction (CCL4 and TAA) were established in a series of pilot experiments by comparing the effect of intraperitoneal (IP) injections vs oral gavage, and constant vs escalating dose protocols on hepatic collagen accumulation. Constant hepatotoxin doses (1.75 ml/kg for CCL4 and 200 mg/kg for TAA) and 3 weekly injections were found to be insufficient to induce significant liver fibrosis in WT C57Bl/6 mice, resulting in a 2-fold increase in liver hydroxyproline (not shown). Both gavage and IP injections of escalating doses of CCL4 led to advanced liver fibrosis in 6 weeks (with a 4-fold increase in liver hydroxyproline, comparable to the rat model 18 ), but the IP route was abandoned due to frequent peritoneal adhesions, accumulation of vehicle (oil), and inflammatory infiltrates in the peritoneal cavity of mice after 6 weeks of treatment. CCL4 was given in mineral oil via oral gavage 3 times a week for up to 12 weeks according to an escalating dose protocol (first dose, ml/kg; week 1 3, 1.75 ml/kg; week 4 6, 2.5 ml/kg; week 7 12, 3.25 ml/kg). Alternatively, fibrosis was induced by escalating the dose of IP TAA for 6 weeks (first dose, 100 mg/kg, week 1 2, 200 mg/kg; week 3 4, 300 mg/kg; week 4 6, 400 mg/kg). Mice were always sacrificed 3 days after the last CCL4 or TAA application, unless otherwise specified. Livers and spleens were weighed at sacrifice and livers snap-frozen for further analysis. Hepatic collagen content determination. Hepatic collagen content was determined as relative hydroxyproline ( g/g liver) in 300 to 400 mg liver samples from 2 different lobes after hydrolysis in 6N HCl for 16 hours at 110 C as described. 18 Total hydroxyproline (mg/whole liver) was calculated based on individual liver weights and the corresponding relative hydroxyproline content. 18,19 Fibrotic matrix stability assessment. ECM stability was assessed biochemically by complete collagen fractionation via serial extractions from snap-frozen livers. Solubility of collagens in the ECM is inversely correlated to the extent of cross-linking. 20 To characterize and quantify the stability of the collagenous ECM, we employed stepwise collagen solubilization from livers using neutral salt (freshly secreted collagens and procollagens), acetic acid (more mature collagens), and acid pepsin (fibrillar, moderately cross-linked collagens) as described by us previously 21,22 and summarized in Figure 2A. Briefly, 1 g snap-frozen liver (3 samples per group, each pooled from pieces of 2 3 individual livers) was homogenized in 20 ml neutral salt buffer (0.5M NaCl, 0.05M Tris, ph 7.5, with Complete Protease Inhibitor; Roche) and incubated at 4 C overnight on a rotary shaker. After centrifugation at 24,000g for 30 minutes, the supernatant was collected for collagen determination (fraction A: neutral salt-soluble collagen). The resulting pellet was extracted with 20 ml 0.5M acetic acid (fraction B: acidsoluble collagen), followed by pepsin (2 mg/ml in 0.5M acetic acid, fraction C: pepsin-soluble collagen). The remaining insoluble fraction D represents mature, highly cross-linked collagen. Each fraction was hydrolyzed in 6N HCl, concentrated 10-fold by evaporation under nitrogen and used to quantify the collagen content via hydroxyproline determination (see Hepatic collagen content determination). Efficient extraction was confirmed by a repeated extraction with fresh buffer, which yielded 2% of collagens extracted in the first step. The percentage of collagen in each fraction was calculated as fraction of the total hydroxyproline determined in all fractions. The recovery of collagens in all fractions was routinely 90% of the total tissue content as determined in parallel samples. Tissue transglutaminase activity assay. Tissue transglutaminase activity was determined in liver homogenates using the biotinamidopentylamine incorporation assay as described in detail previously, 23 with modifications. Briefly, a 96-well plate was coated with N,N-dimethylcasein (Sigma C9801; 10 g/ml in 50 mm Tris, 0.15M NaCl, 5 mm EDTA [ph 7.5]) for 16 hours, 40 C and followed by 3 washing steps with phosphate-buffered saline/0.1% Tween. Human recombinant TG2, expressed and purified as described previously 24 (1 100 ng), or liver homogenate (5 g/well), was added to the reaction buffer

4 May 2011 TISSUE TRANSGLUTAMINASE IN LIVER FIBROSIS 1645 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. (50 mm Tris, 0.15M NaCl, 5 mm CaCl 2, 5 mm dithiothreitol, 0.1% Tween [ph 7.5]), immediately followed by the addition of 4 M biotinamidopentylamin (Pierce Nr 21345) and incubation at room temperature for 2 to 4 hours. Incorporation of biotinamidopentylamine into dimethylcasein was quantified using 50 L horseradish peroxidase conjugated streptavidin (Sigma S 2438, in phosphate-buffered saline/0.1% Tween) and 50 L tetramethylbenzidine substrate and detection at 630 nm. Quantitative Real-Time Reverse- Transcription Polymerase Chain Reaction Relative messenger RNA (mrna) levels were quantified by real-time reverse-transcription polymerase chain reaction on a LightCycler 1.5 instrument (Roche, Mannheim, Germany) using the TaqMan and SYBR Green methodology as described by us in detail. 18,19,25 TaqMan probes (dual-labeled with 5=-FAM and 3=- TAMRA) and primers (summarized in Supplementary Table 1) were designed and validated as described. 18,19,25 Statistical Analyses Data are expressed as mean standard error of mean, and statistical analyses were performed using Microsoft EXCEL (Microsoft, Redmond, WA) and Graph- Pad Prism version 5.00 (GraphPad Software, San Diego, CA). Multiple comparisons were performed by 1-way analysis of variance. Two planned comparisons were performed to each of the control groups: healthy nonfibrotic mice (sham) and mice at peak of fibrosis in fibrosis reversal experiments using the Dunnett s post-test. Differences among selected experimental groups with P values.05 were considered significant. Results Increase in TG2 Expression and Activity Parallels Progression of Experimental Liver Fibrosis in Mice Previous cross-sectional studies indicated an increase in TG2 mrna and activity in humans with liver fibrosis. 12 We further characterized the dynamic changes in TG2 gene expression and activity in relation to both fibrosis stage and activity of fibrogenesis in a model of progressive liver fibrosis due to chronic CCL4 injection in C57Bl6 mice. An optimized protocol of CCL4 administration with an escalating dose regimen (see Materials and Methods) led to robust collagen deposition and progressive fibrosis detectable both biochemically, as an increase in hydroxyproline, and histologically, as an increase in fibrosis stage. Fibrosis was characterized as incipient at week 1 (mean stage F1 according to Metavir, 1.98-fold collagen increase), moderate at week 3 (mean stage F2, 3.44-fold collagen increase), advanced at week 6 (stage F2 F3, 4.56-fold increase in collagen), and early cirrhosis at week 12 (stage F3 F4, 6.21-fold collagen increase) (Figure 1A, B and Table 1). Splenomegaly, an indirect measure of portal hypertension, developed starting at week 6 (spleen weight, mg), reaching mg at 12 weeks, compared to mg in healthy controls (P.0001). TG2 mrna was elevated 3-fold after 1 week and remained elevated 2-fold up to 12 weeks (Figure 1C). Total TG (transamidating) activity was moderately elevated at any time point tested during fibrosis induction (up to 1.5-fold compared to normal controls at week 3, Figure 1E). Moreover, there was a strong correlation of TG2 mrna to commonly used markers of fibrogenic activity (P.001), such as

5 1646 POPOV ET AL GASTROENTEROLOGY Vol. 140, No. 5 Table 1. Changes in Liver and Spleen Weight and Hepatic Collagen Content in C57Bl/6 Mice Treated With an Escalating Dosing Regimen of CCL4 Via Gastric Gavage, as Described in Materials and Methods Group Liver weight, g Spleen weight, mg Relative HYP, g/100 mg Total HYP, g/liver Ctrl (n 6) CCL4, 1 week (n 7) a a a CCL4, 3 weeks (n 7) a a a CCL4, 6 weeks (n 7) a a a a CCL4, 12 weeks (n 6) a a a a NOTE. Data are expressed as mean standard error of mean. Liver and spleen weights were measured at sacrifice and collagen deposition was determined biochemically as relative ( g/g) hydroxyproline (HYP) content in 250 mg liver samples from 2 different lobes. Total HYP (mg/whole liver) was calculated from individual liver weights and respective relative HYP values. CCL4, fibrotic mice treated with carbon tetrachloride for 1, 3, 6, and 12 weeks; Ctrl, nonfibrotic control group (n 6) that received vehicle (mineral oil) only. a P.05 as compared with Ctrl (analysis of variance followed by Dunnett s post-test). transcripts for procollagen 1(I) mrna (the major collagen chain present in scar tissue, r in Pearson test, Figure 1D), transforming growth factor 1 (the major profibrogenic cytokine, r , not shown) and smooth muscle actin (a hepatic stellate cell activation marker, r , not shown). Analysis of Hepatic Fibrotic Matrix In Vivo Reveals Progressive Collagen Stabilization To study dynamic changes in ECM stability in liver fibrosis, we performed a comprehensive analysis of the extractability of collagens from livers along the progression of CCL4-induced hepatic fibrosis. Liver tissue from each group of mice was homogenized and subjected to a series of sequential collagen extractions using neutral salt, acetic acid, and pepsin to solubilize the collagens from the hepatic ECM (Figure 2A). The amount of collagen extracted at each step (as well as the collagen remaining in the insoluble fraction) was directly quantified by hydroxyproline determination after complete hydrolysis of each fraction. In normal murine liver, saltsoluble, and acid-soluble fractions representing newly synthesized collagens amounted to 3.17% and 1.16% of total collagen, respectively. The pepsin-soluble (modestly to moderately cross-linked) fraction represented the vast majority of collagens (91.14%), while 4.53% of collagens were not solubilized by any of the extraction procedures mentioned previously. During fibrosis progression, the salt- and acid-soluble fractions did not change significantly at any time point compared to healthy liver, suggesting a constant proportion of freshly deposited collagen. However, the insoluble collagen fraction increased dramatically with fibrosis progression, from 4.5% in control livers to almost 27% (6-fold) at week 12 of CCL4 administration (Figure 2B). This increase in highly crosslinked, insoluble collagen apparently occurred at the expense of the pepsin-soluble fraction, which decreased from 91% in controls to 70% in mice treated with CCL4 for 12 weeks (Figure 2B). Genetic Deletion of TG2 Decreases Total TG Activity in the Normal and Fibrotic Liver TG2, which is prominently expressed in the liver, was hypothesized to play a role in fibrotic matrix stabilization, thus limiting fibrosis reversibility, 12,13 but it remained to be shown if lack of TG2 (or pharmacologic inhibition of its activity) would lead to (1) reduced fibrosis due to facilitated matrix degradation and removal during fibrogenesis and (2) a less stable fibrotic ECM that would be more susceptible to proteolytic degradation in the recovery (reversal) phase. By using mice deficient in TG2, we showed that the deletion of TG2 was indeed associated with a significant (2-fold) decrease in total TG activity in the normal and fibrotic liver in vivo. Reduced TG activity failed to increase in fibrotic TG2 / livers, indicating that no compensation occurred from other TG isoforms (see Supplementary Figure 1 and Supplementary Material for further details). Therefore, TG2 / mice represent a reliable model to study the functional consequences of deficient TG activity (or its pharmacological inhibition) in fibrotic ECM turnover in the liver, and were used in the subsequent experiments. TG2 Deficiency Does Not Facilitate Collagen Removal During Short-Term Recovery From Hepatotoxin-Induced Liver Fibrosis Next, we addressed the question of how significantly the lack of TG2 expression and activity affects the properties of the fibrotic ECM during spontaneous recovery, and whether this matrix will be more prone to degradation and removal once the profibrogenic stimuli are eliminated. After 6 weeks of CCL4 treatment as established previously (Figure 1), the peak of fibrosis group and the healthy controls (Ctrl, receiving vehicle only) of both TG2 / and TG2 / mice were sacrificed, and the extent of fibrosis evaluated. CCL4 injections were stopped in another group of fibrotic mice, which were allowed to recover ( recovery group) for an additional 4 weeks without any further treatment. WT and TG2 / mice developed advanced bridging fibrosis after 6 weeks

6 May 2011 TISSUE TRANSGLUTAMINASE IN LIVER FIBROSIS 1647 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)orTAA(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)orTAA(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 1(I) [COL1A1]), transforming growth factor 1 (TGF 1) 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 2MG 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). of CCL4 treatment, with TG2 / mice displaying a slightly higher (10%) total collagen content (not significant), as determined by biochemical quantification and histological assessment (Figure 3, Supplementary Table 2), in agreement with histological evidence in an earlier report. 26 This trend of more collagen deposited in the TG2 / mice was confirmed in the mechanistically different model of TAA induced liver fibrosis (a 15% higher

7 1648 POPOV ET AL GASTROENTEROLOGY Vol. 140, No. 5 relative and total collagen content compared to WT mice [not significant]; Supplementary Table 2). After 4 weeks of recovery in both TG2 / and WT mice, inflammatory infiltrates had disappeared almost completely (not shown), and profibrogenic gene expression had normalized (Figure 3C). This was associated with a notable improvement of liver histology, characterized mainly by hepatocyte regeneration and recovery from toxic damage (reduced vacuolization and eosinophilic necrosis) (Figure 3B, E), in accordance with earlier observations by us and others in rats and mice. 13,18 The number of smooth muscle actin positive cells, representing largely activated myofibroblasts, increased dramatically at the peak of fibrosis and returned to nearly normal as early as after 4 weeks of recovery, without significant difference between WT and TG2 / mice and the fibrosis models (Supplementary Figure 2). Notably, there was no decrease in total collagen content in either WT or TG2 / mice recovering from CCL4- or TAA-induced fibrosis for 4 weeks (Figure 3A, D). Lack of TG2 Does Not Facilitate Collagen Removal During Long-Term Recovery From Hepatotoxin-Induced Liver Fibrosis In order to exclude the possibility that 4 weeks of recovery were insufficient to unequivocally demonstrate a lack of advanced fibrosis resolution, we extended the recovery phase up to 36 weeks in mice with CCL4-induced fibrosis. Long-term recovery was accompanied by a significant decrease in relative hepatic (amount per gram of tissue) collagen content beginning at 12 weeks of recovery ( 26% in TG2 / mice at 12 weeks and 23% in WT mice at 24 weeks, compared to the peak of fibrosis). However, when total collagen content was determined factoring in individual liver weights, the reduction was not significant, irrespective of genotype and of the duration of recovery up to 36 weeks (Figure 4C). Thus, the average total collagen content per liver, even after 36 weeks of recovery, was reduced by only 12.7% and 7.4% in WT and TG2 / mice, respectively, not reaching statistical significance in any group. This is in line with our earlier observations in the rat model of TAAinduced fibrosis when livers were examined at 8 weeks of recovery. 18 Therefore, the reduction in relative collagen content was evidently due to hepatocyte regeneration and a subsequent increase in liver mass after cessation of the injury (Figure 4) and not due to true fibrolysis, because the 23% to 26% reduction in relative collagen content was paralleled by a corresponding 18% to 20% increase in liver weights (Figure 4C, Supplementary Table 3). This was confirmed microscopically by a thorough examination of connective tissue staining, which demonstrated the persistence of fibrillar collagen deposits throughout the hepatic parenchyma after 36 weeks of recovery, which were now more loosely associated within the former locations of the fibrotic septa (Figure 4D). Interestingly, a frequent finding was that invading hepatocytes caused dissipation and widening of the fibrotic septa, splitting these into multiple strands of thinner fibrils, as also demonstrated by morphometric analysis (Figure 4E, F). This was evident as early as 4 weeks of fibrosis induction in both genotypes, further increasing up to 36 weeks after CCL4 withdrawal (Figure 4E), and closely resembled features of fibrotic liver remodeling described in advanced human fibrosis. 27 Stability of the Fibrotic Matrix Is Not Affected by the Lack of TG2 During Progression or Recovery Finally, we quantitatively assessed the effect of TG2 deficiency on fibrotic matrix stabilization during progression and recovery from CCL4-induced liver fibrosis using fractional collagen extractions as described here. At none of the several time points of fibrosis progression did collagen extractability differ between WT and TG2 / mice, including the insoluble, highly cross-linked scar collagen. Interestingly, after 36 weeks of recovery, the percentage of insoluble collagen tended to be slightly higher in both WT and TG2 / mice (24.3% and 21.4%, respectively), compared to the peak of fibrosis (19.69% and 16.69%, respectively). This may indicate that the process of ECM stabilization does not stop (or reverse) after elimination of the fibrogenic stimulus, but even continues, although at a much slower pace, even in the absence of TG2 (Figure 5). Additional studies are needed to confirm this observation. Discussion TG2 has been proposed to play an important role in fibrosis due to its (in vitro) activity to cross-link collagen within the fibrotic matrix, thus limiting fibrosis reversibility. In vivo evidence was based on the detection of -( -glutamyl) lysine isopeptide cross-links in fibrotic livers of humans 12 and rodents. 13 In our study, we characterized dynamic changes in ECM (collagen) stability in progressive liver fibrosis and directly addressed the functional contribution of TG2, the predominant hepatic TG isoform, to collagen stabilization during fibrosis progression and fibrosis reversal in vivo using mice with genetic deletion of TG2. Analysis of fibrotic ECM stability via sequential collagen extractions revealed that irreversible collagen stabilization is a characteristic feature of progressive fibrosis, with a striking stage-dependent increase in the pepsin-insoluble fraction, which was accompanied by an increase in TG2 activity and mrna expression. However, although TG2 expression and activity increased during fibrosis progression and strongly correlated with transcript levels of fibrogenic genes, TG2 deficiency had no measurable effect on the stability of the fibrotic ECM or on reversibility of hepatic fibrosis in vivo in 2 hepatotoxin-induced models. Thus, contrary to what was widely believed, TG2-mediated cross-linking does not seem to

8 May 2011 TISSUE TRANSGLUTAMINASE IN LIVER FIBROSIS 1649 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).

9 1650 POPOV ET AL GASTROENTEROLOGY Vol. 140, No. 5 Figure 5. Genetic deletion of TG2 does not affect solubility of collagen in mice without fibrosis, and mice with advanced fibrosis or after longterm 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 acidsoluble 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). functionally contribute to collagen stabilization and irreversibility of fibrosis in the liver. Targeting ECM stability is an attractive strategy to treat fibrosis, and to increase its reversibility once the underlying cause has been eliminated. 3 Although this field is largely unexplored, there are isolated reports supporting a potential benefit of blocking post-translational collagen modifications. Thus, earlier studies suggest that administration of -aminopropionitrile, which irreversibly inhibits lysyl oxidase dependent collagen cross-linking, might mitigate development of cirrhosis, 28 and partially prevent the increase in liver stiffness determined by direct rheometry in fibrotic rats subjected to chronic CCL4 administration. 29 Interestingly, fibrotic matrix stiffness itself, which is believed to depend on collagen cross-linking, has been shown to directly activate primary hepatic myofibroblasts (the major fibrogenic effector cell in the liver), when they are cultured on artificial matrices of increasing stiffness. 30 Moreover, it was demonstrated that mice lacking ADAMTS2, the protease that permits lateral alignment and cross-linking of freshly secreted triple helical collagen to collagen fibrils by cleaving the N-terminal procollagen propeptide from procollagens type I, II, III, and V, had a slower rate of collagen deposition during chronic CCL4 administration and a higher rate of fibrosis reversal after CCL4 withdrawal. 31 These data, coupled with the observation that fibrous septa in AD- AMTS2 / mice were made of thinner and irregular collagen fibers, is consistent with the concept that collagen maturation and stabilization are important therapeutic targets in hepatic fibrosis. In vivo studies on collagen stabilization are difficult, requiring the direct quantification of various collagen cross-links after complete hydrolysis of the liver tissue with multiple internal standards and expensive equipment, 6 and they are hampered by the lack of reagents to detect cross-links in situ. Indeed, we were not able to reliably detect the TG-generated -( -glutamyl) lysine isopeptide in terminal proteolytic digests of normal or fibrotic liver homogenates by high-performance liquid chromatography and mass spectrometry based methods. This is likely due both to the low abundance of this cross-link in vivo and an inhibitory matrix effect in complex biological samples (see Supplementary Figures 3 and 4 and Supplementary Material for details). With regard to TG2-mediated cross-links, erroneous results may have been produced in the past by using commercial antibodies that show unspecific binding in the context of native proteins. 32 We therefore used mice with genetic deletion of TG2 and compromised transamidating activity as the major approach, coupled with quantification of the degree of overall collagen cross-linking using sequential collagen extraction and the model of spontaneous fibrosis reversal as a major functional end point. We clearly demonstrated that progressive collagen cross-linking is a central characteristic during liver fibrosis progression in vivo, which to our knowledge had not been shown directly in models of experimental hepatic fibrosis before. We developed a simple and highly reproducible methodology to assess ECM stability via sequential collagen extractions, which will be useful for future studies on fibrotic matrix stabilization and the evaluation of antifibrotic agents targeting this mechanism. It is plausible that this process, which we show to be TG2-independent, limits the reversibility of hepatic fibrosis, even once the fibrogenic stimuli are eliminated. Surprisingly, no quantitative reduction in total liver collagen was observable in either WT or in TG2 / mice during long-term recovery for up to 36 weeks, indicating that collagen degradation and removal does not occur during the recovery phase. This finding contradicts an earlier report that suggested that mice bearing a mutation in the procollagen I gene, which confers resistance to collagenase, demonstrate a lack of spontaneous collagen degradation after recovery from CCL4-induced fibrosis. 33 Although the aforementioned study reports a 43% decrease in relative hydroxyproline levels in WT controls and impaired hepatocyte proliferation in r/r mice, changes in liver volume were not considered and total hepatic collagen content was not determined. Our present and earlier studies 18,34 indicate that an increase in hepatocyte

10 May 2011 TISSUE TRANSGLUTAMINASE IN LIVER FIBROSIS 1651 mass during regeneration results in changes in relative collagen levels during recovery, even in the absence of collagen degradation. Importantly, 36 weeks of recovery in mice is equivalent to roughly one third of their life span, comparable to 20 to 30 years in humans. From the clinical perspective, because most patients are diagnosed with advanced fibrosis or cirrhosis in their 4 6 th decade, it is tempting to speculate that even if the cause of hepatic fibrosis is eliminated, eg, due to successful pharmacological treatment of hepatitis B or C, fibrolytic therapy that actively reverses advanced fibrosis/early cirrhosis may still be required. Although TG2 deficiency did not facilitate reversal of liver fibrosis, TG2 / -deficient mice seem to be slightly more susceptible to liver inflammation and fibrosis, consistent with an earlier study. 26 One plausible explanation would be the role of TG2 in facilitating apoptosis and phagocytic removal of apoptotic cells, 35,36 which are processes directly implicated in control of both inflammation and fibrosis progression. 37 Indeed, the observed profibrogenic effect of TG2 deficiency that is unrelated to ECM cross-linking is consistent with inefficient macrophage phagocytosis. This may shift the balance toward fibrogenesis by limiting fibrolysis because macrophages secrete fibrolytic MMPs upon apoptotic cell engulfment and play a critical role in (biliary) fibrosis reversal. 34 However, whatever the contribution of these and other activities of TG2 are, our approach clearly demonstrated that overall TG2 does not favor collagen (ECM) crosslinking, nor significantly affect (liver) fibrosis progression or regression. In conclusion, we demonstrate that (1) TG2 is upregulated during fibrogenesis but is dispensable for the stabilization of the fibrotic liver ECM in vivo; (2) the fibrotic matrix formed in the absence of TG2 is not more easily degradable during spontaneous recovery from liver fibrosis than that of WT mice; and (3) TG2-independent irreversible collagen cross-linking is a remarkable feature of progressive hepatic fibrosis, and represents a promising target for novel antifibrotic therapies. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at doi: /j.gastro References 1. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet 2008;371: Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008;134: Popov Y, Schuppan D. Targeting liver fibrosis: strategies for development and validation of antifibrotic therapies. Hepatology 2009;50: Schuppan D, Ruehl M, Somasundaram R, Hahn EG. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis 2001;21: Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol Life Sci 2006;63: Robins SP. Biochemistry and functional significance of collagen cross-linking. Biochem Soc Trans 2007;35: Beninati S, Piacentini M. The transglutaminase family: an overview: minireview article. Amino Acids 2004;26: Collighan RJ, Griffin M. Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications. Amino Acids 2009;36: Kim SY, Jeitner TM, Steinert PM. Transglutaminases in disease. Neurochem Int 2002;40: Elli L, Bergamini CM, Bardella MT, Schuppan D. Transglutaminases in inflammation and fibrosis of the gastrointestinal tract and the liver. Dig Liver Dis 2009;41: Telci D, Griffin M. Tissue transglutaminase (TG2) a wound response enzyme. Front Biosci 2006;11: Grenard P, Bresson-Hadni S, El Alaoui S, Chevallier M, Vuitton DA, Ricard-Blum S. Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis. J Hepatol 2001;35: Issa R, Zhou X, Constandinou CM, et al. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 2004; 126: Jones RA, Kotsakis P, Johnson TS, et al. Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth. Cell Death Differ 2006;13: Zhou X, Jamil A, Nash A, et al. Impaired proteolysis of collagen I inhibits proliferation of hepatic stellate cells: implications for regulation of liver fibrosis. J Biol Chem 2006;281: Schuppan D, Junker Y, Barisani D. Celiac disease: from pathogenesis to novel therapies. Gastroenterology 2009;137: De Laurenzi V, Melino G. Gene disruption of tissue transglutaminase. Mol Cell Biol 2001;21: Popov Y, Patsenker E, Bauer M, Niedobitek E, Schulze-Krebs A, Schuppan D. Halofuginone induces matrix metalloproteinases in rat hepatic stellate cells via activation of p38 and NFkappaB. J Biol Chem 2006;281: Popov Y, Patsenker E, Fickert P, Trauner M, Schuppan D. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary fibrosis via massive dysregulation of pro- and antifibrogenic genes. J Hepatol 2005;43: Oxlund H, Jorgensen PH, Ortoft G, Andreassen TT. Alterations in the cross-links of skin collagen of rats treated with biosynthetic growth hormone. Connect Tissue Res 1991;26: Schuppan D, Cantaluppi MC, Becker J, et al. Undulin, an extracellular matrix glycoprotein associated with collagen fibrils. J Biol Chem 1990;265: Becker J, Schuppan D, Benzian H, et al. Immunohistochemical distribution of collagens types IV, V, and VI and of pro-collagens types I and III in human alveolar bone and dentine. J Histochem Cytochem 1986;34: Dieterich W, Esslinger B, Trapp D, et al. Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease. Gut 2006;55: Dieterich W, Trapp D, Esslinger B, et al. Autoantibodies of patients with coeliac disease are insufficient to block tissue transglutaminase activity. Gut 2003;52: Popov Y, Patsenker E, Stickel F, et al. Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies. J Hepatol 2008;48:

11 1652 POPOV ET AL GASTROENTEROLOGY Vol. 140, No Nardacci R, Lo Iacono O, Ciccosanti F, et al. Transglutaminase type II plays a protective role in hepatic injury. Am J Pathol 2003;162: Wanless IR, Nakashima E, Sherman M. Regression of human cirrhosis. Morphologic features and the genesis of incomplete septal cirrhosis. Arch Pathol Lab Med 2000;124: Fiume L, Favilli G. Inhibition of experimental cirrhosis by carbon tetrachloride following treatment with aminoacetonitrile. Nature 1961;189: Georges PC, Hui JJ, Gombos Z, et al. Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis. Am J Physiol Gastrointest Liver Physiol 2007;293:G1147 G Li Z, Dranoff JA, Chan EP, Uemura M, Sevigny J, Wells RG. Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture. Hepatology 2007;46: Kesteloot F, Desmouliere A, Leclercq I, et al. ADAM metallopeptidase with thrombospondin type 1 motif 2 inactivation reduces the extent and stability of carbon tetrachloride-induced hepatic fibrosis in mice. Hepatology 2007;46: Johnson GV, LeShoure R Jr. Immunoblot analysis reveals that isopeptide antibodies do not specifically recognize the epsilon- (gamma-glutamyl)lysine bonds formed by transglutaminase activity. J Neurosci Methods 2004;134: Issa R, Zhou X, Trim N, et al. Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. FASEB J 2003;17: Popov Y, Sverdlov DY, Bhaskar KR, et al. Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis. Am J Physiol Gastrointest Liver Physiol 2010;298:G323 G Sarang Z, Toth B, Balajthy Z, et al. Some lessons from the tissue transglutaminase knockout mouse. Amino Acids 2009;36: Szondy Z, Sarang Z, Molnar P, et al. Transglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc Natl Acad Sci U S A 2003;100: Canbay A, Higuchi H, Bronk SF, et al. Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis. Gastroenterology 2002;123: Received January 26, Accepted January 13, Reprint requests Address requests for reprints to: Detlef Schuppan, MD, PhD, Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Dana 501, 330 Brookline Avenue, Boston, Massachussetts dschuppa@bidmc. harvard.edu; fax: or to Yury Popov, MD, PhD, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Dana 501, 330 Brookline Avenue, Boston, Massachussetts ypopov@bidmc.harvard.edu; fax: Acknowledgments We would like to thank Jessica Zaks (Beth Israel Deaconess Medical Center) for her excellent technical assistance with the animal experiments, Professor M. Griffin (Nottingham Trent University, UK) for sharing protocols and expert advice for performing terminal proteolytic liver digests for cross-link quantification, Mary Adams (Dana-Farber Cancer Institute, Boston) for help with high-performance liquid chromatography, and Suzanne White (Beth Israel Deaconess Medical Center) for her valuable assistance with histology. Conflicts of interest The authors disclose no conflicts. Funding This work was supported by an appointment grant by the Beth Israel Deaconess Medical Center, and the National Institutes of Health (grants NIH 1 R21 DK A1 and 1 R21 DK A2) to D.S. Y.P. was the recipient of a Sheila Sherlock fellowship of the European Association for the Study of the Liver.