Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation
2013; Elsevier BV; Volume: 183; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2013.03.018
ISSN1525-2191
AutoresKaori Kuramitsu, Deanna Sverdlov, Susan B. Liu, Eva Csizmadia, Linda C. Burkly, Detlef Schuppan, Douglas W. Hanto, Leo E. Otterbein, Yury Popov,
Tópico(s)Organ Transplantation Techniques and Outcomes
ResumoFailure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis. Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis. The liver is the only organ that has the impressive ability to regenerate after injury or surgical resection.1Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2918) Google Scholar Partial hepatectomy (PHx) is the most commonly used model for studying this unique capacity of the liver. After PHx, up to 95% of hepatocytes begin to replicate to compensate for the lost tissue and, in mice, regeneration reaches a maximum of 30 to 60 hours.2Fausto N. Campbell J.S. The role of hepatocytes and oval cells in liver regeneration and repopulation.Mech Dev. 2003; 120: 117-130Crossref PubMed Scopus (564) Google Scholar The remnant liver increases its volume until the regenerated liver mass approaches the original volume. This proliferation of hepatocytes is followed by proliferation of biliary epithelial cells and sinusoidal cells, and full restoration of hepatic architecture and function.1Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2918) Google Scholar The canals of Hering connect the terminal segment of the biliary ductal system with parenchymal hepatocytes.3Roskams T.A. Theise N.D. Balabaud C. Bhagat G. Bhathal P.S. Bioulac-Sage P. Brunt E.M. Crawford J.M. Crosby H.A. Desmet V. Finegold M.J. Geller S.A. Gouw A.S. Hytiroglou P. Knisely A.S. Kojiro M. Lefkowitch J.H. Nakanuma Y. Olynyk J.K. Park Y.N. Portmann B. Saxena R. Scheuer P.J. Strain A.J. Thung S.N. Wanless I.R. West A.B. Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.Hepatology. 2004; 39: 1739-1745Crossref PubMed Scopus (574) Google Scholar, 4Saxena R. Theise N.D. Crawford J.M. Microanatomy of the human liver-exploring the hidden interfaces.Hepatology. 1999; 30: 1339-1346Crossref PubMed Scopus (134) Google Scholar Cells residing in the canals of Hering, called oval cells because of their morphology, function as adult hepatic stem cells. Oval cells express both fetal hepatocyte and biliary cell markers and have the ability to generate both hepatocytes and cholangiocytes,5Sell S. Heterogeneity and plasticity of hepatocyte lineage cells.Hepatology. 2001; 33: 738-750Crossref PubMed Scopus (385) Google Scholar thus considered to be bipotent progenitor cells in adult liver.6Roskams T. Different types of liver progenitor cells and their niches.J Hepatol. 2006; 45: 1-4Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Liver regeneration can occur via two distinct pathways, hepatocyte- and progenitor (oval) cell-mediated. After PHx performed on the healthy liver, hepatocytes are the primary replicating cells responsible for liver regeneration. Although contribution of intrahepatic and extrahepatic (bone marrow) stem cell was proposed, recent and carefully conducted cell fate-tracing studies confirm that normal liver regeneration occurs via mature hepatocyte proliferation.7Malato Y. Naqvi S. Schürmann N. Ng R. Wang B. Zape J. Kay M. Grimm D. Willenbring H. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.J Clin Invest. 2011; 121: 4850-4860Crossref PubMed Scopus (340) Google Scholar Progenitor (oval) cell activation leading to hepatocyte regeneration is not observed during this process.2Fausto N. Campbell J.S. The role of hepatocytes and oval cells in liver regeneration and repopulation.Mech Dev. 2003; 120: 117-130Crossref PubMed Scopus (564) Google Scholar, 7Malato Y. Naqvi S. Schürmann N. Ng R. Wang B. Zape J. Kay M. Grimm D. Willenbring H. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.J Clin Invest. 2011; 121: 4850-4860Crossref PubMed Scopus (340) Google Scholar On the other hand, oval cell proliferation is prominent in some experimental models of liver injury and carcinogenesis induced by Azo dyes, choline deficient and ethionine-containing diets, D-galactosamine, acetylaminofluorene, or CCl4 treatment.8Shafritz D.A. Dabeva M.D. Liver stem cells and model systems for liver repopulation.J Hepatol. 2002; 36: 552-564Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar When hepatectomy is combined with inhibition of mature hepatocyte replication, regeneration occurs primarily via the proliferation of oval cells and their differentiation into hepatocytes.2Fausto N. Campbell J.S. The role of hepatocytes and oval cells in liver regeneration and repopulation.Mech Dev. 2003; 120: 117-130Crossref PubMed Scopus (564) Google Scholar The wound healing response is a series of cellular and molecular events necessary for prompt tissue repair after injury.9Hantash B.M. Zhao L. Knowles J.A. Lorenz H.P. Adult and fetal wound healing.Front Biosci. 2008; 13: 51-61Crossref PubMed Scopus (102) Google Scholar Chronic liver injury often results in hepatic fibrosis, defined by excessive extracellular matrix deposition in periportal areas or in the parenchyma that may progress to cirrhosis with distortion of hepatic architecture, compromised function, and life-threatening complications. Cirrhosis is common end-stage pathology of chronic liver disease of numerous etiologies. Although the mechanisms that lead to the progression of fibrosis, as well as the specific cells, mediators, and transcription factors that contribute to fibrosis progression are increasingly understood,10Friedman S.L. Mechanisms of hepatic fibrogenesis.Gastroenterology. 2008; 134: 1655-1669Abstract Full Text Full Text PDF PubMed Scopus (2209) Google Scholar no clinically proven anti-fibrotic treatment exists.11Popov Y. Schuppan D. Targeting liver fibrosis: strategies for development and validation of antifibrotic therapies.Hepatology. 2009; 50: 1294-1306Crossref PubMed Scopus (260) Google Scholar Hepatic resection is rarely performed in patients with liver cirrhosis, even of Child-Pugh grade A, due to poor outcomes. It is clinically well known that in the setting of advanced fibrosis, liver regeneration is severely impaired,12Llovet J.M. Burroughs A. Bruix J. Hepatocellular carcinoma.Lancet. 2003; 362: 1907-1917Abstract Full Text Full Text PDF PubMed Scopus (3813) Google Scholar but a lack of mechanistic understanding of this phenomenon has severely hampered efforts to improve ability of fibrotic liver to regenerate and permit resection in these patients. To date, there is ample experimental literature focusing on liver regeneration after PHx in normal livers, but fibrotic liver regeneration remains understudied. Here, we describe the detailed characterization of a murine model of PHx of fibrotic liver, which permits further insights into molecular mechanisms of regeneration in the context of fibrotic liver and importantly allows formal preclinical testing of potential regeneration enhancing agents. We demonstrate for the first time that therapeutic targeting of the profibrogenic progenitor (oval) cell response offers a novel approach by which to improve regeneration in fibrotic livers. All animal experiments and procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center (Harvard Medical School, Boston, MA; protocols #189-2006 and #158-2008). C57BL/6 mice (6-week-old males) were purchased from the Jackson Laboratory (Bar Harbor, ME) and used in experiments after 1 week of acclimatization in the animal facility at Beth Israel Deaconess Medical Center. CCl4 and mineral oil were purchased from Sigma-Aldrich (St. Louis, MO). A 70% PHx was performed 3 days after the last dose of CCl4 according to the standard procedure as previously described in mice,13Greene A.K. Puder M. Partial hepatectomy in the mouse: technique and perioperative management.J Invest Surg. 2003; 16: 99-102Crossref PubMed Scopus (146) Google Scholar with pre-established liver fibrosis induced (n = 86) or without (n = 144), described as follows. Briefly, anesthesia was induced by inhalation of isoflurane (2% v/v). After laparotomy, the left and median liver lobes were removed separately, distal to 4-0 silk ties. Body weights and the survival of each mouse were postoperatively recorded every day, and mice were sacrificed at selected time points after surgery. Liver fibrosis was induced in C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME), according to an optimized escalating dose regimen of CCl4 in mineral oil by oral gavage three times per week (Monday, Wednesday, and Friday) for 6 weeks, as previously established.14Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Nonfibrotic controls received mineral oil as a vehicle only. Mice were sacrificed at 1, 2, 3, 5, 10, and 14 days after PHx to access liver regeneration parameters (n = 10 to 21 per time point), essentially as previously described.15Kuramitsu K. Gallo D. Yoon M. Chin B.Y. Csizmadia E. Hanto D.W. Otterbein L.E. Carbon monoxide enhances early liver regeneration in mice after hepatectomy.Hepatology. 2011; 53: 2016-2026Crossref PubMed Scopus (30) Google Scholar Body weights at the time of sacrifice were measured, liver specimens and blood samples were collected, and liver weights were recorded. Resected liver tissues were immediately snap frozen in liquid nitrogen and were used as preresection controls to be compared to postresection parameters in each individual mouse. In initial experiments, both mineral oil-treated mice and untreated mice were used as normal controls (mineral oil-treated mice out of total per time point: day 1, 4 of 10; day 2, 8 of 16; day 3, 2 of 10; day 10, 6 of 16; day 14, 3 of 10). Because none of the general parameters differed among these control groups, as related to liver regeneration [body weight change after surgery, liver regeneration, hepatocyte proliferation, and alanine aminotransferase (ALT)] (levels of ALT, data not shown), the untreated mice were used as normal controls in all subsequent experiments. For progenitor (oval) cell inhibition experiments, mice were dosed intraperitoneally with either 75 μg neutralizing anti–tumor necrosis factor-like weak inducer of apoptosis (TWEAK) monoclonal antibody (clone P2D10, Biogen Idec, Cambridge, MA)16Campbell S. Burkly L.C. Gao H.X. Berman J.W. Su L. Browning B. Zheng T. Schiffer L. Michaelson J.S. Putterman C. Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.J Immunol. 2006; 176: 1889-1898PubMed Google Scholar or 75 μg isotype IgG (mouse IgG2a, clone P1.17) generated from a commercial cell line (ATCC, Manassas, VA). Oval cell proliferation was induced with recombinant TWEAK-Fc fusion protein (TWEAK-Fc, 75 μg per mouse).16Campbell S. Burkly L.C. Gao H.X. Berman J.W. Su L. Browning B. Zheng T. Schiffer L. Michaelson J.S. Putterman C. Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.J Immunol. 2006; 176: 1889-1898PubMed Google Scholar All injections were made three times: on day 0 (4 hours after surgery), on day 2, and on day 4 after resection. All surviving mice were sacrificed and evaluated on day 5. Liver regeneration ratio was calculated as the percentile of liver weight regained after PHx, as previously described.15Kuramitsu K. Gallo D. Yoon M. Chin B.Y. Csizmadia E. Hanto D.W. Otterbein L.E. Carbon monoxide enhances early liver regeneration in mice after hepatectomy.Hepatology. 2011; 53: 2016-2026Crossref PubMed Scopus (30) Google Scholar Hepatocyte replication and oval cell proliferation were studied by immunohistochemistry (IHC). For the hepatocyte proliferation index, the number of total hepatocytes and Ki-67–positive hepatocytes were determined in each mouse liver by counting 10 random periportal fields at ×200 magnification, and calculating the percentage of replicating Ki-67–positive hepatocytes. Progenitor (oval) cell proliferation was assessed by the oval cell–specific marker A6.17Engelhardt N.V. Factor V.M. Yasova A.K. Poltoranina V.S. Baranov V.N. Lasareva M.N. Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes.Differentiation. 1990; 45: 29-37Crossref PubMed Scopus (88) Google Scholar Cells were counted in at least 10 random periportal fields at ×200 magnification for each mouse liver. IHC, H&E, and connective tissue (Sirius Red) staining were performed in formalin-fixed, paraffin-embedded liver sections, as previously described.18Popov Y. Sverdlov D.Y. Bhaskar K.R. Sharma A.K. Millonig G. Patsenker E. Krahenbuhl S. Krahenbuhl L. Schuppan D. Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G323-G334Crossref PubMed Scopus (107) Google Scholar The primary antibodies used in this study are summarized in Table 1. The A6 antibody was a kind gift from Dr. Valentina Factor (NIH, Bethesda, MD). Horseradish peroxidase–conjugated anti-rat or anti-rabbit antibodies were used for detection, and images were captured using Nikon light microscopy. For immunofluorescence staining, frozen liver tissues (5 μm thick) were cut and mounted on glass slides. Primary antibodies were visualized via conjugation to Alexa Fluor 488 and Alexa Fluor 592 antibodies (Molecular Probes, Life Technologies, Eugene, OR). Nuclei were counterstained blue with DAPI. Proliferation of oval cells was assessed by A6 and proliferating cell nuclear antigen (PCNA) double-immunofluorescence and double-positive cells were counted in randomly selected A6-reactive cell clusters at ×200 magnification in 4 to 5 fields per animal (n = 5). For florescence TUNEL staining, an apoptosis detection kit (Roche Diagnostics, Mannheim, Germany) was used by following the manufacturer's protocol. Images were documented with an Axiovert 200M Apotome wide-field microscope and Axiovision software version 4.6 (Zeiss, Thornwood, NY).Table 1List of Primary Antibodies for IHC and ImmunofluorescencePrimary antibodyTypeSourceKi-67 (clone TEC-3)Rat monoclonalDako (Carpinteria, CA)Phospho-histone3Rabbit polyclonalCell Signaling (Danvers, MA)A6Rat monoclonalDr. Valentina Factor (NIH/NCl)PCNARabbit polyclonalSanta Cruz Biotechnology (Dallas, TX)PCNA, proliferating cell nuclear antigen. Open table in a new tab PCNA, proliferating cell nuclear antigen. Relative mRNA levels were quantified in total liver RNA by real-time RT-PCR on a LightCycler 1.5 instrument (Roche Diagnostics) using the TaqMan methodology as we previously described in detail.19Popov 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: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar TaqMan probes (dual-labeled with 5′-FAM and 3′-TAMRA) and primers for major profibrogenic mRNA quantitation were previously designed and extensively validated.14Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 19Popov 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: 1045-1054Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 20Popov Y. Patsenker E. Stickel F. Zaks J. Bhaskar K.R. Niedobitek G. Kolb A. Friess H. Schuppan D. 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: 453-464Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar As a quantitative measure of fibrosis, hepatic collagen deposition was determined biochemically via relative hydroxyproline content (μg/g liver) in 250 to 300 mg liver samples from two different lobes after hydrolysis in 6N HCl for 16 hours at 110°C as previously described.21Popov 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: 15090-15098Crossref PubMed Scopus (93) Google Scholar To assess collagen turnover in the settings of rapid liver mass changes during regeneration, total hepatic hydroxyproline (mg per whole liver) was calculated based on individual liver weights and the corresponding relative hydroxyproline content as previously described.14Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. Lee J. Dieterich W. Melino G. Schuppan D. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 18Popov Y. Sverdlov D.Y. Bhaskar K.R. Sharma A.K. Millonig G. Patsenker E. Krahenbuhl S. Krahenbuhl L. Schuppan D. Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G323-G334Crossref PubMed Scopus (107) Google Scholar Total collagen content in remnant lobes liver at the time of surgery (day 0) was calculated by multiplying relative hydroxyproline content determined in removed lobes during hepatectomy by the weight of the remnant liver estimated from the weight of removed lobes (based on the assumption that 70% of the liver is removed and 30% remains). Thereafter, collagen changes were assayed directly in remnant regenerating liver by determining relative hydroxyproline in 200 to 300 mg sample, and multiplied by remnant weight to determine total hydroxyproline (ug per liver). Serum levels of transforming growth factor beta (TGFβ)1 were assayed using eBioscience Human/Mouse TGF beta1 ELISA kit (#88-8350, eBioscience Inc., San Diego, CA) according to the manufacturer's instructions following additional centrifugation at 10,000 × g for 10 minutes at 4°C to remove platelets. Results are expressed at means ± SEM. Statistical analyses were performed using the Student's t-test, and P values < 0.05 were considered significant. Survival data were analyzed using Kaplan-Meier estimates, and two-way analysis of variance was performed for the comparison between the two groups in the time course. After 6 weeks of CCl4 administration, all mice developed robust liver fibrosis. Total hepatic collagen content increased fourfold compared to control livers (Figure 1A) (P < 0.001), and the degree of fibrosis was characterized as intermediate, based on histological assessment (equivalent to Metavir score F2/F3) (Figure 1B). Three days after the last CCl4 injection, a 70% PHx was performed and general physiological parameters (survival rate, body weight change, and percentage of regenerated liver weight) were compared between fibrotic mice and nonfibrotic controls. After hepatectomy, 90.8% of all nonfibrotic mice survived the surgery, whereas mice with ongoing fibrosis showed a significantly reduced survival rate (68.9%; P < 0.05) (Figure 1C). Fatalities in nonfibrotic mice were earlier (days 1 to 3) than in fibrotic mice (days 3 to 6) after hepatectomy, suggesting that mortality in the fibrotic group was related to compromised liver regeneration rather than to surgical complications. Nonfibrotic mice lost weight steadily after PHx from day 1, peaking at day 3 (−12.4 ± 4.6%), whereas fibrotic mice lost more weight over a longer period of time, peaking at day 4 (−19.3 ± 8.3%; P < 0.001) (Figure 1D). To assess liver regeneration, we monitored restoration of liver weight at various time points after resection. As expected, nonfibrotic PHx mouse liver restored to original weight (38% increase at day 10 compared to day 1). In comparison, the liver weights in fibrotic PHx mice increased by only 11% at day 10 compared to day 1 (Figure 1E) (P < 0.001). These findings suggest that liver regeneration in fibrotic mice was severely compromised, even before cirrhosis is established, closely resembling the poor postresection outcomes in patients with advanced liver fibrosis and cirrhosis.12Llovet J.M. Burroughs A. Bruix J. Hepatocellular carcinoma.Lancet. 2003; 362: 1907-1917Abstract Full Text Full Text PDF PubMed Scopus (3813) Google Scholar Next, we aimed to characterize the sequence of events responsible for deficient liver regeneration in the context of liver fibrosis. PHx induced a sharp increase in serum ALT at postoperative day 1 in both normal and fibrotic mice, but fibrotic mice showed a greater increase (1530 ± 107 IU/L in fibrotic versus 693 ± 166 IU/L in nonfibrotic mice, P < 0.05). Additionally, fibrotic mice showed a second, unexpected peak in ALT levels in the late recovery phase between days 4 and 6, peaking on day 5 (3542 ± 246 IU/L versus 93 ± 65 IU/L in nonfibrotic controls, P < 0.0001) (Figure 2A). Accordingly, serum AST, alkaline phosphatase, and total bilirubin increased in normal mice only transiently at 1 day, but peaked in fibrotic mice at a late day 5 time point and remained elevated at day 10 (Supplemental Table S1). Careful histological examination revealed that at postoperative day 5, 71% of fibrotic mice showed focal eosinophilic lesions in the periportal area consisting of large numbers of dead hepatocytes not found in the fibrotic livers at earlier (days 1 to 3) or later (days 10 to 14) time points and at no point in regenerating nonfibrotic livers (Figure 2C). TUNEL staining revealed a high rate of cell apoptosis, predominantly localizing to areas of focal hepatocyte death, suggesting that hepatocyte cell death occurred via apoptosis (Figure 2C). In fibrotic livers before PHx, TUNEL+ cells were observed in fewer numbers and localized within portal tracts and fibrotic septa (Figure 2D), whereas normal livers were virtually TUNEL negative before and 5 days after hepatectomy. No cholangiocyte death could be detected histologically (neither in bile ducts nor pseudo ducts of normal or fibrotic mice, not shown). After 70% PHx in nonfibrotic livers, organ volume and function was quickly restored via robust replication of almost all mature hepatocytes, as assessed by immunostaining of liver sections for the nuclear proliferation marker Ki-67. In normal livers before surgery, hepatocytes were quiescent. After PHx, hepatocytes began to proliferate, peaking at day 2 postsurgery (79% Ki-67+ hepatocytes) (Figure 2, B and E). As expected in fibrotic livers, the baseline hepatocyte proliferation was elevated before PHx due to ongoing chronic liver injury and hepatocyte regeneration. However, Ki-67 staining in livers of fibrotic mice was severely blunted with only 1.8% and 10.5% of hepatocytes proliferating on days 2 and 3, respectively, compared with 7.3% and 79.1% in normal mice (P < 0.05) (Figure 2, B and E). Moreover, the rate of Ki-67–positive hepatocytes failed to increase at later time points (Figure 2B). These findings suggest that in the context of the fibrotic microenvironment, liver regeneration after PHx is severely compromised, primarily by the failure of hepatocyte replication in early states (days 1 to 3), followed by massive focal hepatocyte death at day 5. When hepatocyte replication is inhibited during regeneration after PHx, the liver restores its mass via the alternative pathway of adult hepatic progenitor (oval) cell activation.2Fausto N. Campbell J.S. The role of hepatocytes and oval cells in liver regeneration and repopulation.Mech Dev. 2003; 120: 117-130Crossref PubMed Scopus (564) Google Scholar We hypothesized that failure of hepatocyte replication and increased cell death after PHx in fibrotic livers may elicit a similar progenitor response, and analyzed the oval cell compartment using the progenitor (oval) cell marker A6. In normal livers, A6-positive staining was observed only within the portal tracts in quiescent (before PHx) and proliferating bile ducts (days 5 and 10 post-PHx). In fibrotic mice before resection, the number of A6-positive cells was markedly increased and could be observed both in the portal area and within fibrotic septa extending into the lobule. After PHx in fibrotic mice, the percentage of A6-positive cells further increased dramatically from day 5 and peaked at day 10 (threefold increase compared to pre-PHx level and 14-fold increase compared to normal liver (Figure 3A and B) (P < 0.05) at days 5 and 10 versus normal liver, returning to pre-PHx levels by day 14. The staining pattern of A6 was associated with scattered spindle-shaped cells and irregular-shaped bile duct-like proliferations (Figure 3A), characteristic of the pseudoducts formed by proliferating progenitor (oval) cells. These cells also stained positive for the alternative progenitor marker pan-cytokeratin (not shown). Sham surgery alone did not elicit progenitor response (Supplemental Figure S1). Interestingly, increase in circulating TGFβ1 levels in fibrotic (but not normal) mice serum coincided with hepatocyte death and progenitor (oval) cell expansion at 5 days (Figure 3C). Evidently, the expansion of progenitor (oval) cells in fibrotic livers occurred via active proliferation, because nuclei of A6-immunoreactive progenitor cells were frequently positive for the proliferation marker PCNA, as demonstrated by double-immunostaining (Figure 3D). At day 5 post-PHx, at least one PCNA-positive nucleus was detectable in 52% of randomly selected A6-reactive cell clusters, with an average of 9.93 ± 2.3% PCNA-positive cells in A6-positive cell population. Given that the mobilization of hepatic progenitors is clinically associated with progressive liver fibrosis,22Lowes K.N. Brennan B.A. Yeoh G.C. Olynyk J.K. Oval cell numbers in human chronic liver diseases are directly related to disease severity.Am J Pathol. 1999; 154: 537-541Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 23Clouston A.D. Powell E.E. Walsh M.J. Richardson M.M. Demetris A.J. Jonsson J.R. Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis.Hepatology. 2005; 41: 809-818Crossref PubMed Scopus (307) Google
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