Ballooned hepatocytes, undead cells, sonic hedgehog, and vitamin E: therapeutic implications for nonalcoholic steatohepatitis.
Without question, nonalcoholic steatohepatitis (NASH) has emerged as a substantial public health problem. The complex and intertwined cellular and molecular mechanisms culminating in the pathophysiology of this disease process remain only partially understood, and therapeutic options remain suboptimal. Lipotoxic hepatocyte injury is a cardinal feature of NASH pathogenesis,1, 2 and ballooned hepatocytes are a prominent histopathological feature of lipotoxic hepatic injury. In fact, the magnitude of ballooned hepatocytes correlates with disease severity,3 and semiquantitation of hepatocyte ballooning is used to calculate the nonalcoholic fatty liver disease activity score (NAS).4 Diehl et al. made a seminal insight when they discovered that ballooned hepatocytes generate sonic hedgehog (Shh), a ligand of the hedgehog-signaling pathway, which promotes hepatic fibrogenesis.5, 6 These data provided mechanistic insight into a mechanism contributing to hepatic fibrogenesis in NASH. However, several relevant questions remain. What is the ballooned hepatocyte, and why does it generate Shh? Does NASH-targeted therapy alter the number of ballooned hepatocytes in NASH? What is the spectrum of Shh signaling in NASH, and is hedgehog signaling inhibition a strategic pharmacologic strategy for NASH? Despite being a hallmark of NASH, little is known about ballooned hepatocytes. They are posited to represent a special form of "cell degeneration" associated with cellular enlargement, loss of cellular polarity, an abundance of intracellular lipids and oxidized phospholipids and are further characterized by loss of keratin 8/18 and accumulation of ubiquitinated proteins.7 However, these latter characteristics have not been extensively validated and are based on immunohistochemistry (IHC), a semiquantitative technique fraught with concerns regarding sensitivity and specificity. Better characterization of these cells is needed. The original observation by Diehl et al. that ballooned cells produce Shh not only shed light on liver injury, but also on the potential pathogenesis of these cells. In Drosophila melanogaster, cells in which the cell death program has been initiated, but cannot be executed, exist as "undead cells" and secrete factors, including Shh, to aid in tissue repair and healing.8 Although ballooned hepatocytes have yet to be generated in vitro, Kakisaka et al. modeled the undead cell concept by treating hepatocytes deficient in caspase 9 (a protease essential for execution of the mitochondrial pathway of cell apoptosis9) with toxic saturated free fatty acids (FFAs).10 Lipotoxicity in these cells was associated with c-Jun N-terminal kinase (JNK) activation, which, in turn, induced Shh expression in the absence of cell death (Fig. 1). Intriguingly, ballooned hepatocytes in a small number of NASH specimens also exhibit reduced expression of caspase 9, perhaps explaining their persistence despite lipotoxic insults. In the Kakisaka et al. study, Shh also served as an autocrine survival factor for the undead cell, raising the testable hypothesis that inhibition of hedgehog signaling would lead to deletion of ballooned hepatocytes. The ballooned hepatocyte may be analogous to the undead cell characterized in D. melanogaster, a concept requiring further investigation as does the role of JNK signaling in promoting Shh expression in this cellular phenotype. Finally, to ultimately solve the conundrum of how important this minor cell population of ballooned hepatocytes and their production of Shh are in the progression of NASH, a specific depletion of the ballooned hepatocytes in vivo by a genetic approach will be required. Schematic overview of hedgehog pathway activation in NASH. Simplified illustration demonstrates that JNK activation by toxic lipids leads to Shh production in ballooned hepatocytes. Released Shh acts through the autocrine pathway as a survival factor for "undead" ballooned hepatocytes and, through the paracrine mechanism, induces fibroblast differentiation into extracellular matrix–producing myofibroblasts. Vitamin E therapy decreases the amount of Shh in NASH liver by an unknown mechanism. The current study by Guy et al. in this issue of Hepatology tested the hypothesis that NASH regression is associated with decreased activity of the hedgehog-signaling pathway. The investigators evaluated liver biopsies and clinical data from a recent National Institute of Diabetes and Digestive and Kidney Diseases–sponsored clinical trial, PIVENS (PIoglitazone, Vitamin E for Non-alcoholic Steatohepatitis). The trial demonstrated that, compared to placebo, therapy with vitamin E, but not pioglitazone, improved steatosis, lobular inflammation, and hepatocellular ballooning, but not fibrosis, in adult patients with aggressive NASH who did not have diabetes or cirrhosis.11 For the current study, the investigators evaluated samples from the vitamin E and placebo treatment group. They unfortunately excluded the pioglitazone-treated group from their analysis, which could have served as an interesting control, because pioglitazone lacked beneficial effects in NASH patients. In both the placebo and vitamin E groups, the investigators were able to demonstrate that a reduction in the number of Shh-positive hepatocytes over time directly correlates with an improvement in serum alanine aminotransferase and aspartate aminotransferase values, biomarkers of liver injury. Moreover, in the whole cohort, responders (patients with an improvement in NAS scores) displayed a greater decrease in Shh-positive cells, as compared to nonresponders. Interestingly, vitamin E therapy decreased the number of Shh-positive hepatocytes in both responders and nonresponders. When comparing both groups of nonresponders, patients from the vitamin E study arm revealed a greater improvement in liver enzymes and lower number of Shh-positive cells. Collectively, improvement in NASH was associated with decreased hedgehog pathway activity, as assessed by number of Shh-positive hepatocytes. One mechanistic interpretation of these data is that vitamin E, as an antioxidant, prevents oxidative stress associated with JNK activation, resulting in inhibition of Shh autocrine survival signaling with deletion of ballooned hepatocytes (Fig. 1). The fact that vitamin E therapy also reduced Shh-positive hepatocytes in nonresponders suggests that other unrelated mechanisms also contribute to tissue injury in this disease. Hedgehog pathway signaling is initiated by its ligands, such as Shh. In the canonical signaling cascade, these ligands bind to the plasma membrane receptor, patched, which leads to disinhibition of another plasma membrane receptor, smoothened. Interruption of this basal smoothened suppression allows for nuclear translocation of glioma-associated oncogene (Gli) transcription factors. This canonical signaling pathway has been best established in mammalian cells with cilia.12 For example, Guy et al. used IHC to identify Gli2-positive cells in the liver, strong evidence for canonical hedgehog-signaling pathway activation.13 Besides the canonical pathway, noncanonical signaling cascades have been described, which do not require cilia. This noncanonical pathway also requires smoothened, but does not involve Gli-mediated transcription responses, rather smoothened functions as a G-protein-coupled receptor in this signaling pathway.14 In animal models of NASH, hedgehog signaling promotes hepatic fibrogenesis (Fig. 1).5 Generally, fibroblasts, which express cilia, are canonical hedgehog pathway-responsive cells. However, the decreased hyperactivation of the hedgehog pathway in the current study was not accompanied by a significant decrease in fibrosis. Rather, the strongest correlation was found between hedgehog activation and hepatocellular injury. This raises the question of hedgehog signaling in hepatocytes. Indeed, it has been previously reported that hepatocytes express smoothened and other components of the hedgehog-signaling cascade.15 Importantly, smoothened inhibition by vismodegib, an U.S. Food and Drug Administration (FDA)-approved drug for the treatment of basal cell carcinoma,16 attenuated liver injury in a mouse model of NASH.15 Smoothened inhibition in this study prevented tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor up-regulation and TRAIL receptor-mediated injury. The effect of smoothened inhibition on TRAIL receptor expression in hepatocytes was reproduced in vitro using FFA treatment. Future research endeavors are required to determine whether hedgehog signaling in hepatocytes by a noncanonical cascade contributes to liver injury in NASH and/or whether the effect of smoothened inhibition on TRAIL receptor expression is a direct or indirect effect. With increasing prevalence of obesity, NASH has become a growing health problem in the Western world. At the moment, there is no effective therapy for NASH, except for lifestyle modifications, which are difficult to obtain and sustain. Therefore, there is a general agreement that pharmacological therapy for NASH will be required for selected individuals, and current research seeks to identify potential therapeutic targets. Given the substantial body of evidence supporting the importance of hedgehog pathway hyperactivation in NASH, clinical trials using hedgehog-signaling pathway inhibitors, several of which have already been approved by the FDA for other indications, might take us a step closer to effective therapy for NASH. Perhaps even intermittent (once-weekly) therapy would be sufficient to delete ballooned hepatocytes, thereby reducing disease progression. Petra Hirsova, Ph.D.Gregory J. Gores, M.D. Division of Gastroenterology and HepatologyMayo ClinicRochester, MN
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