Cynthia A. Moylan

Disparities in Liver Transplantation Before and After Introduction of the MELD Score

CONTEXT In February 2002, the allocation system for liver transplantation became based on the Model for End-Stage Liver Disease (MELD) score. Before MELD, black patients were more likely to die or become too sick to undergo liver transplantation compared with white patients. Little information exists regarding sex and access to liver transplantation. OBJECTIVE To determine the association between race, sex, and liver transplantation following introduction of the MELD system. DESIGN, SETTING, AND PATIENTS A retrospective cohort of black and white patients (> or = 18 years) registered on the United Network for Organ Sharing liver transplantation waiting list between January 1, 1996, and December 31, 2000 (pre-MELD cohort, n = 21 895) and between February 28, 2002, and March 31, 2006 (post-MELD cohort, n = 23 793). MAIN OUTCOME MEASURES Association between race, sex, and receipt of a liver transplant. Separate multivariable analyses evaluated cohorts within each period to identify predictors of time to death and the odds of dying or receiving liver transplantation within 3 years of listing. Patients with hepatocellular carcinoma were analyzed separately. RESULTS Black patients were younger (mean [SD], 49.2 [10.7] vs 52.4 [9.2] years; P < .001) and sicker (MELD score at listing: median [interquartile range], 16 [12-22] vs 14 [11-19]; P < .001) than white patients on the waiting list for both periods. In the pre-MELD cohort, black patients were more likely to die or become too sick for liver transplantation than white patients (27.0% vs 21.7%) within 3 years of registering on the waiting list (odds ratio [OR], 1.51; 95% confidence interval (CI), 1.15-1.98; P = .003). In the post-MELD cohort, black race was no longer associated with increased likelihood of death or becoming too sick for liver transplantation (26.5% vs 22.0%, respectively; OR, 0.96; 95% CI, 0.74-1.26; P = .76). Black patients were also less likely to receive a liver transplant than white patients within 3 years of registering on the waiting list pre-MELD (61.6% vs 66.9%; OR, 0.75; 95% CI, 0.59-0.97; P = .03), whereas post-MELD, race was no longer significantly associated with receipt of a liver transplant (47.5% vs 45.5%, respectively; OR, 1.04; 95% CI, 0.84-1.28; P = .75). Women were more likely than men to die or become too sick for liver transplantation post-MELD (23.7% vs 21.4%; OR, 1.30; 95% CI, 1.08-1.47; P = .003) vs pre-MELD (22.4% vs 21.9%; OR, 1.08; 95% CI, 0.91-1.26; P = .37). Similarly, women were less likely than men to receive a liver transplant within 3 years both pre-MELD (64.8% vs 67.6%; OR, 0.80; 95% CI, 0.70-0.92; P = .002) and post-MELD (39.9% vs 48.7%; OR, 0.70; 95% CI, 0.62-0.79; P < .001). CONCLUSION Following introduction of the MELD score to the liver transplantation allocation system, race was no longer associated with receipt of a liver transplant or death on the waiting list, but disparities based on sex remain.

Hedgehog pathway activation and epithelial-to-mesenchymal transitions during myofibroblastic transformation of rat hepatic cells in culture and cirrhosis

Myofibroblastic hepatic stellate cells (MF-HSC) are derived from quiescent hepatic stellate cells (Q-HSC). Q-HSC express certain epithelial cell markers and have been reported to form junctional complexes similar to epithelial cells. We have shown that Hedgehog (Hh) signaling plays a key role in HSC growth. Because Hh ligands regulate epithelial-to-mesenchymal transition (EMT), we determined whether Q-HSC express EMT markers and then assessed whether these markers change as Q-HSC transition into MF-HSC and whether the process is modulated by Hh signaling. Q-HSC were isolated from healthy livers and cultured to promote myofibroblastic transition. Changes in mRNA and protein expression of epithelial and mesenchymal markers, Hh ligands, and target genes were monitored in HSC treated with and without cyclopamine (an Hh inhibitor). Studies were repeated in primary human HSC and clonally derived HSC from a cirrhotic rat. Q-HSC activation in vitro (culture) and in vivo (CCl(4)-induced cirrhosis) resulted in decreased expression of Hh-interacting protein (Hhip, an Hh antagonist), the EMT inhibitors bone morphogenic protein (BMP-7) and inhibitor of differentiation (Id2), the adherens junction component E-cadherin, and epithelial keratins 7 and 19 and increased expression of Gli2 (an Hh target gene) and mesenchymal markers, including the mesenchyme-associated transcription factors Lhx2 and Msx2, the myofibroblast marker alpha-smooth muscle actin, and matrix molecules such as collagen. Cyclopamine reverted myofibroblastic transition, reducing mesenchymal gene expression while increasing epithelial markers in rodent and human HSC. We conclude that Hh signaling plays a key role in transition of Q-HSC into MF-HSC. Our findings suggest that Q-HSC are capable of transitioning between epithelial and mesenchymal fates.

Relationship Between Methylome and Transcriptome in Patients With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Cirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting noninvasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression. METHODS We collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0-1 (mild) and F3-4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome. RESULTS Clinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P < .05). Methylation at fibroblast growth factor receptor 2, methionine adenosyl methyltransferase 1A, and caspase 1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, and genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and underexpressed. CONCLUSIONS Functionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH‐related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild‐type (WT) mice, Patched‐deficient (Ptc+/−) (overly active Hh signaling) mice, and OPN‐deficient mice before and after feeding methionine and choline–deficient (MCD) diets to induce NASH‐related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN‐deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up‐regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up‐regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte‐derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh‐regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011)

Hedgehog Signaling Antagonist Promotes Regression of Both Liver Fibrosis and Hepatocellular Carcinoma in a Murine Model of Primary Liver Cancer

Objective Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2−/− mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC. Methods Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2−/− mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging. Results Unlike controls, Mdr2−/− mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality. Conclusions Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.

Leptin promotes the myofibroblastic phenotype in hepatic stellate cells by activating the hedgehog pathway

Trans-differentiation of quiescent hepatic stellate cells (Q-HSCs), which exhibit epithelial and adipocytic features, into myofibroblastic-HSC (MF-HSCs) is a key event in liver fibrosis. Culture models demonstrated that Hedgehog (Hh) pathway activation is required for transition of epithelioid/adipocytic Q-HSCs into MF-HSCs. Hh signaling inhibits adiposity and promotes epithelial-to-mesenchymal transitions (EMTs). Leptin (anti-adipogenic, pro-EMT factor) promotes HSC trans-differentiation and liver fibrosis, suggesting that the pathways may interact to modulate cell fate. This study aimed to determine whether leptin activates Hh signaling and whether this is required for the fibrogenic effects of leptin. Cultures of primary HSCs from lean and fa/fa rats with an inherited ObRb defect were examined. Inhibitors of PI3K/Akt, JAK/STAT, and Hh signaling were used to delineate how ObRb activation influenced Hh signaling and HSC trans-differentiation. Fibrogenesis was compared in wild type and db/db mice (impaired ObRb function) to assess the profibrotic role of leptin. The results demonstrate that leptin-ObR interactions activate Hh signaling with the latter necessary to promote trans-differentiation. Leptin-related increases in Hh signaling required ObR induction of PI3K/Akt, which was sufficient for leptin to repress the epithelioid/adipocytic program. Leptin-mediated induction of JAK/STAT was required for mesenchymal gene expression. Leptin-ObRb interactions were not necessary for HSC trans-differentiation to occur in vitro or in vivo but are important because liver fibrogenesis was attenuated in db/db mice. These findings reveal that leptin activates Hh signaling to alter gene expression programs that control cell fate and have important implications for liver fibrosis and other leptin-regulated processes involving EMTs, including development, obesity, and cancer metastasis.

Hepatic Gene Expression Profiles Differentiate Presymptomatic Patients With Mild Versus Severe Nonalcoholic Fatty Liver Disease

Clinicians rely upon the severity of liver fibrosis to segregate patients with well‐compensated nonalcoholic fatty liver disease (NAFLD) into subpopulations at high‐ versus low‐risk for eventual liver‐related morbidity and mortality. We compared hepatic gene expression profiles in high‐ and low‐risk NAFLD patients to identify processes that distinguish the two groups and hence might be novel biomarkers or treatment targets. Microarray analysis was used to characterize gene expression in percutaneous liver biopsies from low‐risk, “mild” NAFLD patients (fibrosis stage 0‐1; n = 40) and high‐risk, “severe” NAFLD patients (fibrosis stage 3‐4; n = 32). Findings were validated in a second, independent cohort and confirmed by real‐time polymerase chain reaction and immunohistochemistry (IHC). As a group, patients at risk for bad NAFLD outcomes had significantly worse liver injury and more advanced fibrosis (severe NAFLD) than clinically indistinguishable NAFLD patients with a good prognosis (mild NAFLD). A 64‐gene profile reproducibly differentiated severe NAFLD from mild NAFLD, and a 20‐gene subset within this profile correlated with NAFLD severity, independent of other factors known to influence NAFLD progression. Multiple genes involved with tissue repair/regeneration and certain metabolism‐related genes were induced in severe NAFLD. Ingenuity Pathway Analysis and IHC confirmed deregulation of metabolic and regenerative pathways in severe NAFLD and revealed overlap among the gene expression patterns of severe NAFLD, cardiovascular disease, and cancer. Conclusion: By demonstrating specific metabolic and repair pathways that are differentially activated in livers with severe NAFLD, gene profiling identified novel targets that can be exploited to improve diagnosis and treatment of patients who are at greatest risk for NAFLD‐related morbidity and mortality. (Hepatology 2014;59:471–482)

Hedgehog Controls Hepatic Stellate Cell Fate by Regulating Metabolism

BACKGROUND & AIMS The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism. METHODS Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1α (HIF1α) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity. RESULTS Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1α. Inhibitors of Hh signaling, HIF1α, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh. CONCLUSIONS Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.

Viral Factors Induce Hedgehog Pathway Activation in Humans with Viral Hepatitis, Cirrhosis, and Hepatocellular Carcinoma

Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCCs) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCCs develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarciongenesis in chronic viral hepatitis. Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hh-responsive cell types in liver biopsies from 45 patients with chronic HBV or HCV. Hh signaling was then manipulated in cultured liver cells to directly assess the impact of Hh activity in relevant cell types. We found increased hepatic expression of Hh ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC (ie, liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells) were Hh responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC. Inhibiting pathway activity in Hh-responsive target cells reduced fibrogenesis, angiogenesis, and growth. In conclusion, HBV/HCV infection increases hepatocyte production of Hh ligands and expands the types of Hh-responsive cells that promote liver fibrosis and cancer.

Activation of Rac1 promotes hedgehog-mediated acquisition of the myofibroblastic phenotype in rat and human hepatic stellate cells.

Hepatic accumulation of myofibroblastic hepatic stellate cells (MF‐HSCs) is pivotal in the pathogenesis of cirrhosis. Two events are necessary for MF‐HSCs to accumulate in damaged livers: transition of resident, quiescent hepatic stellate cells (Q‐HSCs) to MF‐HSCs and expansion of MF‐HSC numbers through increased proliferation and/or reduced apoptosis. In this study, we identified two novel mediators of MF‐HSC accumulation: Ras‐related C3 botulinum toxin substrate 1 (Rac1) and Hedgehog (Hh). It is unclear whether Rac1 and Hh interact to regulate the accumulation of MF‐HSCs. We evaluated the hypothesis that Rac1 promotes activation of the Hh pathway, thereby stimulating signals that promote transition of Q‐HSCs into MF‐HSCs and enhance the viability of MF‐HSCs. Using both in vitro and in vivo model systems, Rac1 activity was manipulated through adenoviral vector‐mediated delivery of constitutively active or dominant‐negative rac1. Rac1‐transgenic mice with targeted myofibroblast expression of a mutated human rac1 transgene that produces constitutively active Rac1 were also examined. Results in all models demonstrated that activating Rac1 in HSC enhanced Hh signaling, promoted acquisition/maintenance of the MF‐HSC phenotype, increased MF‐HSC viability, and exacerbated fibrogenesis. Conversely, inhibiting Rac1 with dominant‐negative rac1 reversed these effects in all systems examined. Pharmacologic manipulation of Hh signaling demonstrated that profibrogenic actions of Rac1 were mediated by its ability to activate Hh pathway‐dependent mechanisms that stimulated myofibroblastic transition of HSCs and enhanced MF‐HSC viability. Conclusion: These findings demonstrate that interactions between Rac1 and the Hh pathway control the size of MF‐HSC populations and have important implications for the pathogenesis of cirrhosis. HEPATOLOGY 2010