Potential conflict of interest: Drs. Pollard and Charlton own stock in Vertex.
Nonalcoholic fatty liver disease (NAFLD) is a potentially progressive liver disease that culminates in cirrhosis. Cirrhosis occurs more often in individuals with nonalcoholic steatohepatitis (NASH) than in those with steatosis (nonalcoholic fatty liver [NAFL]). The difference between NAFL and NASH is the extent of hepatocyte apoptosis, which is more extensive in NASH. Because phagocytosis of apoptotic cells activates hepatic stellate cells (HSCs), we examined the hypothesis that a pan-caspase inhibitor, VX-166, would reduce progression of fibrosis in a mouse model of NASH. Male db/db mice were fed methionine/choline-deficient (MCD) diets to induce NASH and liver fibrosis. Mice were gavaged once daily with either the pan-caspase inhibitor VX-166 (6 mg/kg/d; Vertex, Abingdon, UK) or vehicle only and sacrificed at 4 or 8 weeks. Treatment with an MCD diet increased alanine aminotransferase (ALT), caspase-3 activity, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL)-positive cells, NASH, and fibrosis. Treatment of MCD-fed mice with VX-166 decreased active caspase-3, TUNEL-positive cells, and triglyceride content (P < 0.05). However, ALT levels were similar in VX-166–treated mice and vehicle-treated controls. Histological findings also confirmed that both groups had comparable liver injury (NAFLD activity score ≥6). Nevertheless, VX-166–treated MCD-fed mice demonstrated decreased α-smooth muscle actin expression (4 weeks, P < 0.05; 8 weeks, P < 0.005) and had reduced hepatic levels of collagen 1α1 messenger RNA (8 weeks, P < 0.05). Hydroxyproline content and Sirius red staining of VX-166–treated livers confirmed decreases in fibrosis. Conclusion: Inhibiting hepatic apoptosis suppresses the development of fibrosis in mice with NASH. Beneficial effects on liver fibrosis were associated with reductions in hepatic steatosis, but occurred without obvious improvement in liver injury. These findings are consistent with evidence that apoptosis triggers HSC activation and liver fibrosis and suggest that caspase inhibitors may be useful as an antifibrotic NASH therapy. (HEPATOLOGY 2009.)
Nonalcoholic fatty liver disease (NAFLD) is a potentially progressive liver disease that culminates in cirrhosis in some individuals. Cirrhosis occurs more often in individuals with nonalcoholic steatohepatitis (NASH) than in those with simple steatosis (nonalcoholic fatty liver [NAFL]).1 A major difference between NAFL and NASH is the extent of hepatocyte apoptosis, which is much more severe in NASH than NAFL.2
It has been demonstrated that hepatocellular apoptosis is a critical mechanism that contributes not only to progression of NAFLD, but also to progression of many other liver diseases, including viral hepatitis, Wilson's disease, cholestatic liver disease, and alcohol-induced injury.3 In all of those injuries, hepatocyte apoptosis has been recognized as a major contributing mechanism for fibrogenesis and cirrhosis.4, 5 Apoptotic bodies are phagocytosed by adjacent cells, and it has been shown that the phagocytosis of apoptotic bodies by quiescent hepatic stellate cells (HSCs) is one of the mechanisms that promote their activation to a myofibroblastic phenotype that produces collagen type I, the major collagen that accumulates in cirrhosis.6
Apoptosis is an ATP-dependent and programmed type of cell death that is mediated by the activation of caspases, intracellular proteases that allow cleavage of protein substrates, causing the disassembly and death of the target cell.7 This process can occur in several different ways; in the liver, however, apoptosis is predominantly mediated by activation of Fas death receptors, which are often overexpressed in injured and dying hepatocytes.8 In NASH, Fas-mediated hepatocyte apoptosis has been demonstrated to be significantly up-regulated and associated with advanced stages of the disease.2 This has also been demonstrated in a murine model of NASH induced by a methionine/choline-deficient (MCD) diet.9–11
The mammalian caspase family consists of 14 different proteases that share similar composition characterized by two large and two small subunits that associate in a tetramer.7 Similarly to other proteases, caspases are synthesized as zymogens that undergo proteolytic cleavage in order to exert their activity. When active, all caspases specifically recognize a 4– or 5–amino acid sequence containing an aspartic acid residue on the target protein and cleave at that location. Caspases themselves have to be cleaved at similar aspartate residues to become active.12 Thus, activation of caspases requires caspase activity. Because of this characteristic, it has been possible to design pan-caspase inhibitors that broadly inhibit all caspases. Such pan-caspase inhibitors not only inhibit cellular apoptosis, but also block proteolytic cleavage of certain interleukin (IL) molecules, including IL-1β and IL-18. Both of these interleukins are cleaved by caspase-1 to generate smaller, biologically active proinflammatory cytokines that have been implicated in liver injury pathogenesis.7 Hence, pan-caspase inhibitors have been proposed as attractive therapeutic agents for many types of liver disease, including NASH.
To date, however, these agents have been evaluated mostly in models of acute liver injury, leaving lingering concerns about their efficacy and safety in more chronic types of liver disease. These issue merit consideration because many surviving liver cells in chronically injured livers have up-regulated a variety of antiapoptotic defenses,13 and thus, further pharmacologic inhibition of caspase activity might provide relatively little benefit. Indeed, caspase inhibition in the context of ongoing liver injury might actually be detrimental by limiting the appropriate clearance of premalignant cells and/or myofibroblastic stellate cells. If such effects occur, pan-caspase inhibitors could potentially exacerbate the risk for bad outcomes of chronic liver injury, including cirrhosis and hepatocellular carcinoma. Therefore, in this study, we examined the hypothesis that treatment with a pharmacological pan-caspase inhibitor, VX-166,14 would reduce progression of fibrosis in a mouse model of NASH that has been proven to result in steatohepatitis and liver fibrosis in the context of chronic obesity and type 2 diabetes.10 Our first aim was to determine whether VX-166 reduced or increased liver injury by blocking cellular apoptosis. Our second objective was to determine whether the pan-caspase inhibitor reduced or increased activation of collagen producing myofibroblastic HSCs and/or hepatic fibrogenesis.
Animal Studies and Dietary Model of Steatohepatitis.
Seventy male C57BL/6 obese and diabetic db/db (stock no. 699) and five wild-type C57BL/6 (stock no. 664) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in a temperature- and light-controlled facility. Animals were age-matched and used at approximately 10 weeks of age.
To induce NASH and liver fibrosis, 35 db/db mice were fed an MCD diet. Control mice (n = 35, db/db) were permitted ad libitum consumption of water and standard rodent food. In each group, 25 mice were gavaged once daily with the pan-caspase inhibitor VX-166 (6 mg/kg/d) in polyethylene glycol supplemented with vitamin E, and the remaining 10 mice received polyethylene glycol and vitamin E only. In each group, 12 of the mice were sacrificed at 4 weeks and the remaining mice at 8 weeks (see Supplemental Material). All animal care and procedures performed were approved by the Duke University Medical Center Institutional Animal Care and Use Committee.
Histological and Immunohistochemical Analysis.
Hematoxylin-eosin was performed to assess general histology. Sirius red was used to evaluate liver fibrosis. Standard immunohistochemical analysis with citrate antigen retrieval was performed to localize expression of active caspase-3. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay (Boehringer Mannheim, Mannheim, Germany) was performed according to the manufacturer's suggestions. Immunohistochemical quantification for nitrotyrosine, α-smooth muscle actin (α-SMA), and active caspase-3 was assessed using MetaView (Universal Imaging, Downingtown, PA) (see Supplemental Material). To obtain statistical significance, at least five ×200 magnification random-field images were taken per slide, and at least five animals per group were scored (n = 5).
RNA and Protein Analysis.
RNA was extracted and analyzed by way of quantitative reverse-transcription polymerase chain reaction (QRT-PCR) as described.15 For protein analysis, standard western blot analysis was performed (see Supplemental Material).
Serum and Tissue Analysis.
Serum alanine aminotransferase (ALT) levels were measured using commercially available assay (Biotron Diagnostics; Hemet, CA). Hepatic lipid content was analyzed for total triglycerides (Biotron Diagnostics; Hemet, CA), cholesterol (Amplex Red Cholesterol Assay Kit, Invitrogen), and nonessential fatty acids (NEFAs) (Wako Pure Chemicals Industries, Richmond, VA). All procedures were performed as per manufacturer's instructions.
Hepatic hydroxyproline content was quantified colorimetrically in flash-frozen liver samples according with a previously described method.16 Concentrations were calculated from a standard curve prepared with high-purity hydroxyproline (Sigma-Aldrich) and expressed as milligrams of hydroxyproline per gram of liver.
Results are expressed as the mean ± standard error of the mean (SEM). Significance was established using the Student t test and analysis of variance when appropriate. Differences were considered significant at P < 0.05.
Treatment with VX-166 Was Well-Tolerated and Improved Diet-Induced Steatosis in Obese db/db Mice.
db/db Mice were obese, weighing almost twice as much as age- and sex-matched wild-type mice at the end of the 8-week experiment, regardless of whether they were gavaged daily with vehicle or vehicle plus VX-166. Although mice that were fed MCD diets lost 20% to 30% of their body weight during this time period, the degree of weight loss was not influenced by treatment with VX-166. Also, despite significant weight loss, mice in both MCD diet-fed groups remained significantly more obese than wild-type control mice (Supporting Fig. 1).
Although obese, chow-fed db/db mice had only mild hepatic steatosis, as assessed either histologically (Fig. 1A) or by triglyceride content (Fig. 1D), MCD diets dramatically induced hepatic fat accumulation, increasing hepatic content of triglycerides about 40-fold by 4 weeks (Fig. 1B,D), and NEFAs at both 4 and 8 weeks (Fig. 1F). MCD diets had no effect on total liver cholesterol at 4 weeks, and significantly lowered it at 8 weeks (Fig. 1E). Hepatic triglyceride content also decreased after 8 weeks of MCD diet, but still remained about 15-fold greater than that of chow-fed db/db controls (Fig. 1C,D). Treatment with VX-166 significantly reduced hepatic triglycerides at 4 weeks (Fig. 1B,D), but not at 8 weeks (Fig. 1C,D). It also generally reduced total liver cholesterol in MCD diet-treated animals (Fig. 1E), but significantly increased liver NEFAs at 8 weeks (Fig. 1F).
Treatment with VX-166 Did Not Improve Diet-Induced Liver Injury in db/db Mice.
Serum ALT levels in chow-fed db/db mice were about two-fold higher than in chow-fed wild-type C57Bl6 mice at 4 weeks, and they increased further at 8 weeks (Fig. 2A). In contrast, feeding db/db mice MCD diets caused significant increases in serum ALT, raising values more than 10-fold above levels observed in wild-type C57Bl6 mice. Treatment with VX-166 did not significantly impact ALT levels in db/db mice, regardless of whether they were fed chow or MCD diet. Thus, feeding MCD diets to db/db mice induced steatohepatitis, and treating such mice with VX-166 did not improve ALT, a serum marker of liver injury, despite reducing hepatic steatosis.
To further investigate the effects of treatment on liver injury, hematoxylin-eosin–stained liver sections were carefully reviewed for features of steatohepatitis.17 MCD diets increased histological parameters of liver injury as assessed by NAFLD activity score. VX-166 treatment did not improve MCD diet–related increases in hepatic inflammation, ballooning, or overall NAFLD activity score (Table 1), confirming the apparent lack of benefit of the pan-caspase inhibitor on liver injury caused by diet-induced NASH.
Table 1. Histological Analysis of Steatosis, Inflammation, Ballooning, and Overall NAFLD Activity Score in MCD- and VX-166–Treated db/db and Wild-Type Mice
C57BL/6 Obese db/db Mice
Chow 4-8 Weeks
Chow + VX 4 Weeks
Chow + VX 8 Weeks
MCD 4 Weeks
MCD 8 Weeks
MCD + VX 4 Weeks
MCD + VX 8 Weeks
Abbreviation: NAS, NAFLD activity score.
Similarities in net liver injury in VX-166–treated mice and controls were unanticipated because pan-caspase inhibition was predicted to inhibit production of proinflammatory cytokines and block intracellular signaling that triggers mitochondrial release of reactive oxygen species to cause oxidative liver damage. Therefore, we assessed the effect of VX-166 treatment on hepatic production of tumor necrosis factor α (TNF-α) and accumulation of nitrotyrosine adducts. MCD diets increased hepatic TNF-α messenger RNA (mRNA) levels in control mice at both 4 and 8 weeks, and treatment with VX-166 significantly attenuated diet-related increases in inflammatory cytokine production at both time points (Fig. 2B,C). VX-166 also significantly reduced MCD diet-induced accumulation of nitrotyrosine adducts (Fig. 2D-F). Therefore, treatment with the pan-caspase inhibitor failed to ameliorate MCD diet–induced steatohepatitis despite inhibiting TNF-α production and reducing formation of nitrotyrosine adducts in liver tissue.
VX-166 Retained Its Pan-Caspase Inhibitory Activity During Long-Term Treatment.
To determine whether the drug retained its caspase inhibitory activity when administered according to our study protocol, activated caspase-3 was assessed by performing immunohistochemistry and morphometric analysis. Results in all of the various groups of db/db mice were greater than those in wild-type C57BL/6 mice, in which fewer than one liver cell per ×40 field was noted to contain activated caspase-3 (data not shown). Chow-fed db/db mice had only rare liver cells stained, suggesting that those mice also had very low levels of active caspase-3 (Fig. 3A,D and inset). In contrast, the hepatic activity of caspase-3 was substantially increased by feeding db/db mice MCD diets for either 4 or 8 weeks (Fig. 3B,D). Of note, treatment with VX-166 reproducibly reduced the numbers of liver cells with caspase-3 activity at both time points (Fig. 3C,D).
To further evaluate the apparent efficacy of VX-166 for inhibiting caspase-3 actvity, western blot analysis of whole liver tissues was performed. Immunoblotting demonstrated that the MCD diet treatment increased hepatic accumulation of the caspase-3 cleavage product at 4 weeks, but not at 8 weeks. Treatment with VX-166 significantly decreased levels of activated caspase-3 at both time points, however (lowering levels of the caspase-3 cleavage product by nearly two-fold at 4 weeks and by 50% at 8 weeks [both P < 0.05]) (Fig. 3E and 3F, respectively). Thus, through immunohistochemical analysis and immunoblot assessment, the lowest levels of caspase-3 activity were observed in MCD-fed db/db mice that received VX-166.
To further validate the activity of the putative pan-caspase inhibitor, VX-166, we next examined activation of caspase-1. This caspase is involved in cytokine activation during liver injury and is required for proteolytic cleavage of IL-1β and IL-18, two injury-related, pro-inflammatory cytokines. Western blot analysis of IL-1β preprotein (≈31 kDa) and its smaller, biologically active caspase-1 cleavage product (≈17 kDa) (Fig. 4A,B), as well as the caspase-1 cleavage product of IL-18 (≈20 kDa) (Figs. 4C,D), demonstrated that MCD diets increased hepatic accumulation of both truncated IL-1β and IL-18, and that VX-166 treatment significantly abrogated both processes.
Taken together with the caspase-3 results, these findings indicate that the failure of VX-166 to reduce MCD diet–related liver injury cannot be attributed to a loss of its pan-caspase activity. Rather, the data suggest that caspase-independent mechanisms of liver injury increased to offset any beneficial effects of pan-caspase inhibition. To further investigate this possibility, TUNEL staining was performed. In liver, TUNEL staining marks cells that have been killed by both apoptotic and nonapoptotic mechanisms. MCD diets increased cell death by nearly 25-fold at both 4 and 8 weeks (Fig. 5A,B). Although treatment with VX-166 reduced the number of TUNEL-positive cells by 50% (Fig. 5B), many dead cells were still apparent (Fig. 5A, inset). Therefore, effective inhibition of caspase activation by VX-166 was not sufficient to abrogate liver cell death in mice with MCD diet–induced steatohepatitis, suggesting that nonapoptotic mechanisms might have mediated liver injury in the VX-166–treated groups.
Treatment with VX-166 Inhibited Hepatic Fibrogenesis in MCD Diet-Fed db/db Mice.
Increased apoptosis distinguishes NASH from NAFL, and this is thought to contribute to the increased risk for liver fibrosis in NASH, because phagocytosis of apoptotic bodies promotes myofibroblastic transformation of HSCs. Hence, it was conceivable that the reduced apoptotic activity in VX-166–treated mice might have inhibited activation of HSCs, resulting in less liver fibrosis despite no net improvement in MCD diet–related liver injury. To evaluate this concept, hepatic accumulation of α-SMA, a marker of myofibroblastic HSCs, was examined by way of western blot analysis. As shown in Fig. 6A, treatment with VX-166 reduced α-SMA content in MCD diet–fed db/db mice, causing nearly two-fold reductions in α-SMA protein levels at 4 weeks (P < 0.05) and three-fold at 8 weeks (P < 0.005).
Further immunohistochemical analysis revealed that α-SMA predominantly accumulated in areas of lobular inflammation following MCD diets (Fig. 6B,E). Additionally, MCD diets induced the chicken wire pattern of pericellular/sinusoidal α-SMA staining that is typical of steatohepatitis-related fibrosis in human NASH (Fig. 6E, inset). Following VX166 administration, α-SMA staining decreased (Fig. 6C), particularly around liver cells and along hepatic sinusoids (Fig. 6F). Morphometry confirmed general reductions in α-SMA accumulation after VX166 treatment at both 4 and 8 weeks (Fig. 6D).
To determine if reduced accumulation of α-SMA was accompanied by changes in liver fibrosis, liver sections were stained with Sirius Red, and deposition of collagen fibrils was analyzed (Fig. 7A-D). Chow-fed db/db mice and chow-fed control mice had negligible (and comparable) collagen deposition. MCD diets increased collagen deposition in db/db mice, particularly around hepatocytes and along hepatic sinusoids. Treatment with VX-166 improved pericellular and sinusoidal fibrosis in MCD diet–fed db/db mice. These changes were most notable after 8 weeks of MCD diet exposure, and they were paralleled by improvements in collagen 1α1 mRNA expression, as assessed by way of QRT-PCR analysis (Fig. 7E). Indeed, collagen 1α1 mRNA expression was significantly (P <0.05) reduced at 8 weeks in MCD diet-fed db/db mice that were treated with VX-166. Biochemical assessment of hepatic collagen by way of hydroxyproline assay confirmed the effects of treatment on hepatic collagen content in the MCD diet–fed db/db mice (Fig. 7F). Treatment of such mice with VX-166 reduced collagen accumulation back toward levels that were observed in chow-fed db/db mice. Therefore, inhibiting hepatic apoptosis by treating obese mice with this pan-caspase inhibitor reduced NASH-related fibrogenesis, despite exerting no net benefit on liver injury per se.
In NAFLD, as in other types of chronic liver disease, liver-related morbidity and mortality are greatest in patients who become cirrhotic.18 Although patients with NASH are much more likely to develop cirrhosis than patients with simple hepatic steatosis (NAFL), cirrhosis is not an inevitable consequence of NASH. Therefore, considerable efforts have been devoted to identify and treat individuals with NASH who are at greatest risk for disease progression. That work led to the realization that hepatic accumulation of apoptotic bodies19 and α-SMA–expressing myofibroblastic cells20 were useful predictors of subsequent hepatic fibrosis in patients with NASH. Phagocytosis of apoptotic bodies has been shown to activate HSCs to a myofibroblastic phenotype,6 suggesting that treatments that inhibit hepatocyte apoptosis, such as caspase inhibitors, might prevent progressive fibrogenesis in NASH. On the other hand, because chronic exposure to proapoptotic stresses occurs in NASH, many surviving hepatocytes have up-regulated antiapoptotic defenses.18 Hence, further pharmacologic inhibition of caspases may provide little additional benefit. In addition, blocking caspase activity for an extended period of time in the context of chronic liver injury might interfere with clearance of malignant liver cells and/or myofibroblastic HSCs, thereby enhancing the risk for liver cancer and/or cirrhosis. Until the current studies, these issues had not been directly examined in NASH, leaving questions about the efficacy and safety of these agents.
The present study demonstrates that prolonged treatment with the pan-caspase inhibitor VX-166 failed to improve net liver injury in db/db mice with MCD diet–induced NASH. Although the agent reduced hepatic activity of both initiator and effector caspases and decreased hepatic expression of TNF-α, diet-induced steatosis, and accumulation of nitrotyrosine adducts in liver tissue, it was not sufficient to completely abrogate liver cell death and did not improve various other histologic parameters of liver injury or decrease serum levels of ALT in this mouse model of NASH. Thus, genetically obese mice with diet-induced NASH responded very differently to pan-caspase inhibition than previously healthy lean mice that were subjected to bile duct ligation to induce acute cholestatic liver injury.21 Treatment of the latter mice with a pan-caspase inhibitor significantly improved the histologic features of cholestatic liver injury and dramatically reduced serum ALT values. Further research will be required to determine whether these important differences simply reflect inherent differences in the specific pharmacologic agents or the doses and durations of treatment that were used in the two studies, as opposed to general differences in the relative significance of caspase-dependent and caspase-independent mechanisms in chronic versus subacute liver injury, and/or more specific differences in the relative importance of such mechanisms in NASH versus cholestatic liver disease.
Interestingly, however, despite eliciting very different effects on liver injury, both pan-caspase inhibitors improved hepatic fibrogenesis. Canbay et al.21 reported that treatment of bile duct–ligated mice with the pan-caspase inhibitor IDN-6556 for 3 days demonstrated significant reductions in hepatic expression of transforming growth factor-β and collagen 1α1 mRNA, and 10-day treatment resulted in significant improvement in liver fibrosis as assessed by Sirius red staining. In the current study, MCD diet–fed db/db mice that were treated for 4 or 8 weeks with VX-166 also demonstrated inhibition of collagen gene expression and reduced accumulation of Sirius Red–stained fibrils. Assessment of hepatic hydroxyproline content was also performed in our study, and results provided additional confirmatory biochemical evidence that treatment with VX-166 decreased hepatic fibrogenesis. Whether or not prolonged treatment with VX-166 would be equally efficacious in inhibiting hepatic fibrogenesis in other models of NASH merits investigation, because MCD diets cause weight loss and improve insulin resistance even in genetically obese and diabetic db/db mice, and most humans with NASH are obese and insulin-resistant. In addition, in the present study, VX-166 was administered in conjunction with the NASH-inducing diet, leaving unresolved questions about its potential efficacy for improving liver fibrosis in individuals with well-established fatty liver damage. Such work will be particularly important because liver histology indicated that inhibiting hepatic caspase activity in obese db/db mice with diet-induced NASH did not improve hepatic necroinflammation, and the latter is generally believed to promote liver fibrosis.8
In any case, the current findings raise important new questions about the role of caspase-dependent mechanisms in hepatic injury and fibrogenesis. In Drosophila, activation of initiator and effector caspases trigger distinct signaling mechanisms in dying cells that permit those cells to generate paracrine factors that ultimately provoke compensatory proliferation in neighboring viable cells.22 For example, effector caspase activation in flies increases production of morphogens that have been shown to promote the proliferation and viability of mammalian myofibroblasts.23 In the present study, treatment with VX-166 inhibited activation of caspase-3, a major effector caspase in hepatocytes, and reduced hepatic accumulation of cells expressing the myofibroblast marker, α-SMA. Thus, it is conceivable that the mechanism of hepatocyte death (i.e., apoptosis versus necrosis or some other type of nonapoptotic death), rather than simply the degree/extent of hepatocyte loss, modulates hepatic responses to liver injury, with apoptotic cell death providing a particularly robust signal for fibrogenic repair. Hence, targeted inhibition of effector caspases might provide a novel therapeutic approach to prevent liver fibrosis.