Hepatology
Abstract
Focal adhesion kinase (FAK) is an important mediator of extracellular matrix-integrin mechano-signal transduction that regulates cell motility, survival, and proliferation. As such, FAK is being investigated as a potential therapeutic target for malignant and fibrotic diseases, and numerous clinical trials of FAK inhibitors are underway. The function of FAK in non-malignant non-motile epithelial cells is not well understood. We previously showed that hepatocytes demonstrated activated FAK near stiff collagen tracts in fibrotic liver. In this study, we examined the role of liver epithelial FAK by inducing fibrotic liver disease in mice with liver epithelial FAK deficiency. We found that mice that lack FAK in liver epithelial cells develop more severe liver injury and worse fibrosis as compared to controls. Increased fibrosis in liver epithelial FAK-deficient mice is linked to the activation of several pro-fibrotic pathways, including the hedgehog-smoothened pathway. FAK-deficient hepatocytes produce increased Indian hedgehog in a manner dependent on matrix stiffness. Furthermore, expression of the hedgehog receptor, smoothened, is increased in macrophages and biliary cells of hepatocyte-specific FAK-deficient fibrotic liver. These results indicate that liver epithelial FAK has important regulatory roles in the response to liver injury and progression of fibrosis.
Authors
Yun Weng, Tyler J. Lieberthal, Vivian X. Zhou, Maya Lopez-Ichikawa, Manuel Armas-Phan, Tristan K. Bond, Miya C. Yoshida, Won-Tak Choi, Tammy T. Chang
Abstract
Obesity predisposes to cancer and a virtual universality of nonalcoholic fatty liver disease (NAFLD). However, the impact of hepatic steatosis on liver metastasis is enigmatic. We find that while control mice were relatively resistant to hepatic metastasis, those which were lipodystrophic or obese, with NAFLD, had a dramatic increase in breast cancer and melanoma liver metastases. NAFLD promotes liver metastasis by reciprocal activation initiated by tumor-induced triglyceride lipolysis in juxtaposed hepatocytes. The lipolytic products are transferred to cancer cells via fatty acid transporter protein 1, where they are metabolized by mitochondrial oxidation to promote tumor growth. The histology of human liver metastasis indicated the same occurs in humans. Furthermore, comparison of isolates of normal and fatty liver established that steatotic lipids had enhanced tumor-stimulating capacity. Normalization of glucose metabolism by metformin did not reduce steatosis-induced metastasis, establishing the process is not mediated by the metabolic syndrome. Alternatively, eradication of NAFLD in lipodystrophic mice by adipose tissue transplantation reduced breast cancer metastasis to that of control mice, indicating the steatosis-induced predisposition is reversible.
Authors
Yongjia Li, Xinming Su, Nidhi Rohatgi, Yan Zhang, Jonathan R. Brestoff, Kooresh I. Shoghi, Yalin Xu, Clay F. Semenkovich, Charles A. Harris, Lindsay L. Peterson, Katherine N. Weibaecher, Steven L. Teitelbaum, Wei Zou
Abstract
Orphan nuclear receptor estrogen-related receptor (ERR)γ stimulates bile acid production, however, the role and the regulatory mechanism of ERRγ in cholestatic liver disease are largely unknown. This study identifies that Sirt6 is a deacetylase of ERRγ and suggests a novel mechanism by which Sirt6 activation alleviates cholestatic liver damage and fibrosis through regulating ERRγ. We observed that hepatic expression of Sirt6 is repressed while that of ERRγ is upregulated in murine cholestasis models. Hepatocyte-specific Sirt6 knockout mice were more severely injured following a bile duct ligation (BDL) compared to wild-type mice and adenoviral re-expression of Sirt6 reversed liver damage and fibrosis as demonstrated by biochemical and histological analyses. Mechanistically, Sirt6 deacetylated ERRγ, thereby destabilized ERRγ and inhibited its transcriptional activity. Elimination of hepatic ERRγ using Ad-shERRγ abolished the deleterious effects of Sirt6 deficiency, while ERRγ overexpression aggravated cholestatic liver injury. Administration of a Sirt6 deacetylase activator prevented BDL-induced liver damage and fibrosis. In patients with cholestasis, Sirt6 expression was decreased while total- and acetylated-ERRγ levels were increased, confirming negative regulation of ERRγ by Sirt6. Thus, Sirt6 activation represents a new therapeutic strategy for treating cholestatic liver injury.
Authors
Lihua Hao, In Hyuk Bang, Jie Wang, Yuancheng Mao, Jae Do Yang, Soon-Young Na, Jeong Kon Seo, Hueng-Sik Choi, Eun Ju Bae, Byung-Hyun Park
Abstract
Nonalcoholic fatty liver disease (NAFLD) is a common comorbidity among people living with HIV with a more aggressive course than in the general population. In a recent randomized placebo-controlled trial, we demonstrated that the growth hormone-releasing hormone analogue tesamorelin reduced liver fat and prevented fibrosis progression in HIV-associated NAFLD over one year. As such, tesamorelin is the first strategy that has shown to be effective against NAFLD among the HIV population. The current study leveraged paired liver biopsy specimens from this trial to identify hepatic gene pathways that are differentially modulated by tesamorelin versus placebo. Using Gene Set Enrichment Analysis (GSEA), we found that tesamorelin increased hepatic expression of hallmark gene sets involved in oxidative phosphorylation and decreased hepatic expression of gene sets contributing to inflammation, tissue repair, and cell division. Tesamorelin also reciprocally up- and downregulated curated gene sets associated with favorable and poor hepatocellular carcinoma prognosis, respectively. Notably, among tesamorelin-treated participants, these changes in hepatic expression correlated with improved fibrosis-related gene score. Our findings inform our knowledge of the biology of growth hormone action on the liver and provide a mechanistic basis for the observed clinical effects of tesamorelin on the liver.
Authors
Lindsay T. Fourman, James M. Billingsley, George Agyapong, Shannan J. Ho Sui, Meghan N. Feldpausch, Julia Purdy, Isabel Zheng, Chelsea S. Pan, Kathleen E. Corey, Martin Torriani, David E. Kleiner, Colleen M. Hadigan, Takara L. Stanley, Raymond T. Chung, Steven K. Grinspoon
Abstract
The α-1-antitrypsin (or alpha-1-antitrypsin, A1AT) Z variant is the primary cause of severe A1AT deficiency and forms polymeric chains that aggregate in the endoplasmic reticulum of hepatocytes. Around 2%–5% of Europeans are heterozygous for the Z and WT M allele, and there is evidence of increased risk of liver disease when compared with MM A1AT individuals. We have shown that Z and M A1AT can copolymerize in cell models, but there has been no direct observation of heteropolymer formation in vivo. To this end, we developed a monoclonal antibody (mAb2H2) that specifically binds to M in preference to Z A1AT, localized its epitope using crystallography to a region perturbed by the Z (Glu342Lys) substitution, and used Fab fragments to label polymers isolated from an MZ heterozygote liver explant. Glu342 is critical to the affinity of mAb2H2, since it also recognized the mild S-deficiency variant (Glu264Val) present in circulating polymers from SZ heterozygotes. Negative-stain electron microscopy of the Fab2H2-labeled liver polymers revealed that M comprises around 6% of the polymer subunits in the MZ liver sample. These data demonstrate that Z A1AT can form heteropolymers with polymerization-inert variants in vivo with implications for liver disease in heterozygous individuals.
Authors
Mattia Laffranchi, Emma L.K. Elliston, Elena Miranda, Juan Perez, Riccardo Ronzoni, Alistair M. Jagger, Nina Heyer-Chauhan, Mark L. Brantly, Annamaria Fra, David A. Lomas, James A. Irving
Abstract
Alcoholic liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting hepatocytes to release proinflammatory mitochondrial DNA (mtDNA)-enriched extracellular vesicles (EVs). The aim of this study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol-induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the highest hepatic upregulation of metallothionein 1/2 (Mt1/2) which encode two most potent antioxidant proteins. Genetic deletion of the Mt1/2 gene aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress and ASK1/p38 activation in the liver. Inhibition or genetic deletion of the Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, reactive oxygen species levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.
Authors
Jing Ma, Haixia Cao, Robim M. Rodrigues, Mingjiang Xu, Tianyi Ren, Yong He, Seonghwan Hwang, Dechun Feng, Ruixue Ren, Peixin Yang, Suthat Liangpunsakul, Jian Sun, Bin Gao
Abstract
Extramedullary hematopoietic cells are present in the liver of normal neonates in the first few days of life and persist in infants with biliary atresia. Based on a previous report that liver genes are enriched by erythroid pathways, we examined the liver gene expression pattern at diagnosis and found the top five enriched pathways are related to erythrocyte pathobiology in children who survived with the native liver beyond 2 years of age. Using immunostaining, anti-CD71 antibodies identified CD71+ erythroid cells among extramedullary hematopoietic cells in the livers at the time of diagnosis. In mechanistic experiments, the preemptive antibody depletion of hepatic CD71+ erythroid cells in neonatal mice rendered them resistant to rotavirus (RRV)-induced biliary atresia. The depletion of CD71+ erythroid cells increased the number of effector lymphocytes and delayed the RRV infection of livers and extrahepatic bile ducts. In co-culture experiments, CD71+ erythroid cells suppressed the activation of hepatic mononuclear cells. These data uncover an immunoregulatory role for CD71+ erythroid cells in the neonatal liver.
Authors
Li Yang, Pranavkumar Shivakumar, Jeremy M. Kinder, Sing Sing Way, Bryan Donnelly, Reena Mourya, Zhenhua Luo, Jorge A. Bezerra
Abstract
Autosomal co-dominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria and inflammation, leading to fibrosis, cirrhosis and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first generation intravenously-administered RNA interference (RNAi) therapeutic ARC-AAT and then with next generation subcutaneously-administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria and autophagosomes in hepatocytes were evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in Phase 2/3 clinical trials.
Authors
Christine I. Wooddell, Keith Blomenkamp, Ryan M. Peterson, Vladimir M. Subbotin, Christian Schwabe, James Hamilton, Qili Chu, Dawn R. Christianson, Julia O. Hegge, John Kolbe, Holly L. Hamilton, Maria F. Branca-Afrazi, Bruce D. Given, David L. Lewis, Edward Gane, Steven B. Kanner, Jeffrey H. Teckman
Abstract
Carriers of the hydroxysteroid 17-β dehydrogenase 13 (HSD17B13) gene variant (rs72613567:TA) have a reduced risk of NASH and cirrhosis but not steatosis. We determined its effect on liver histology, lipidome, and transcriptome using ultra performance liquid chromatography-mass spectrometry and RNA-seq. In carriers and noncarriers of the gene variant, we also measured pathways of hepatic fatty acids (de novo lipogenesis [DNL] and adipose tissue lipolysis [ATL] using 2H2O and 2H-glycerol) and insulin sensitivity using 3H-glucose and euglycemic-hyperinsulinemic clamp) and plasma cytokines. Carriers and noncarriers had similar age, sex and BMI. Fibrosis was significantly less frequent while phospholipids, but not other lipids, were enriched in the liver in carriers compared with noncarriers. Expression of 274 genes was altered in carriers compared with noncarriers, consisting predominantly of downregulated inflammation-related gene sets. Plasma IL-6 concentrations were lower, but DNL, ATL and hepatic insulin sensitivity were similar between the groups. In conclusion, carriers of the HSD17B13 variant have decreased fibrosis and expression of inflammation-related genes but increased phospholipids in the liver. These changes are not secondary to steatosis, DNL, ATL, or hepatic insulin sensitivity. The increase in phospholipids and decrease in fibrosis are opposite to features of choline-deficient models of liver disease and suggest HSD17B13 as an attractive therapeutic target.
Authors
Panu K. Luukkonen, Taru Tukiainen, Anne Juuti, Henna Sammalkorpi, P.A. Nidhina Haridas, Onni Niemelä, Johanna Arola, Marju Orho-Melander, Antti Hakkarainen, Petri T. Kovanen, Om Dwivedi, Leif Groop, Leanne Hodson, Amalia Gastaldelli, Tuulia Hyötyläinen, Matej Orešič, Hannele Yki-Järvinen
Abstract
Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal–regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.
Authors
Susanne Schuster-Gaul, Lukas Jonathan Geisler, Matthew D. McGeough, Casey D. Johnson, Anna Zagorska, Li Li, Alexander Wree, Vivian Barry, Igor Mikaelian, Lily J. Jih, Bettina G. Papouchado, Grant Budas, Hal M. Hoffman, Ariel E. Feldstein
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