Microbiome and Metabolome Restoration After Administration of Fecal Microbiota, Live-jslm (REBYOTA®) for Preventing Recurrent Clostridioides difficile Infection.

BACKGROUND: Microbiota-based treatments are effective in preventing recurrent Clostridioides difficile infection (rCDI). Fecal microbiota, live-jslm (REBYOTA®; RBL, previously RBX2660) was shown to prevent rCDI in a phase 3, randomized, double-blinded placebo controlled clinical trial (PUNCH™ CD3). METHODS: Stool samples from participants in PUNCH™ CD3 who received a single blinded dose of rectally administered RBL or placebo were sequenced to determine microbial community composition and calculate the Microbiome Health Index for post-antibiotic dysbiosis (MHI-A). The composition of bile acids (BAs) in the same samples was quantified by liquid chromatography mass spectrometry. Relationships between BA composition and microbiota community structure and correlations with treatment outcomes were assessed. RESULTS: Before administration, Gammaproteobacteria and Bacilli dominated the microbiota community and primary BAs were more prevalent than secondary BAs. Clinical success after administration correlated with shifts to predominantly Bacteroidia and Clostridia, a significant increase in MHI-A, and a shift from primary to secondary BAs. Several microbiota and BA changes were more extensive in RBL-treated responders compared to placebo-treated responders, and microbiota changes correlated with BA changes. CONCLUSIONS: Clinical response and RBL administration were associated with significant restoration of microbiota and BA composition. CLINICAL TRIALS REGISTRATION: NCT03244644.

Molecular MR Imaging of Renal Fibrogenesis in Mice.

BACKGROUND: In most CKDs, lysyl oxidase oxidation of collagen forms allysine side chains, which then form stable crosslinks. We hypothesized that MRI with the allysine-targeted probe Gd-oxyamine (OA) could be used to measure this process and noninvasively detect renal fibrosis. METHODS: Two mouse models were used: hereditary nephritis in Col4a3-deficient mice (Alport model) and a glomerulonephritis model, nephrotoxic nephritis (NTN). MRI measured the difference in kidney relaxation rate, ΔR1, after intravenous Gd-OA administration. Renal tissue was collected for biochemical and histological analysis. RESULTS: ΔR1 was increased in the renal cortex of NTN mice and in both the cortex and the medulla of Alport mice. Ex vivo tissue analyses showed increased collagen and Gd-OA levels in fibrotic renal tissues and a high correlation between tissue collagen and ΔR1. CONCLUSIONS: Magnetic resonance imaging using Gd-OA is potentially a valuable tool for detecting and staging renal fibrogenesis.

pH Dependence of a GPR4 Selective Antagonist Hampers Its Therapeutic Potential.

Inflammatory bowel disease (IBD) is characterized by chronic mucosal inflammation of the gastrointestinal tract and is associated with extracellular acidification of mucosal tissue. Several extracellular pH-sensing receptors, including G protein-coupled receptor 4 (GPR4), play an important role in the regulation of inflammatory and immune responses, and GPR4 deficiency has been shown to be protective in IBD animal models. To confirm the therapeutic potential of GPR4 antagonism in IBD, we tested Compound 13, a selective GPR4 antagonist, in the interleukin 10-/- mouse model of colitis. Despite good exposures and albeit there was a trend toward improvement for a few readouts, Compound 13 treatment did not improve colitis in this model, and there were no signs of target engagement. Interestingly, Compound 13 behaved as an "orthosteric" antagonist, i.e., its potency was pH dependent and mostly inactive at pH levels lower than 6.8 with preferential binding to the inactive conformation of GPR4. Mutagenesis studies confirmed Compound 13 likely binds to the conserved orthosteric binding site in G protein-coupled receptors, where a histidine sits in GPR4 likely preventing Compound 13 binding when protonated in acidic conditions. While the exact mucosal pH in the human disease and relevant IBD mice models is unknown, it is well established that the degree of acidosis is positively correlated with the degree of inflammation, suggesting Compound 13 is not an ideal tool to study the role of GPR4 in moderate to severe inflammatory conditions. SIGNIFICANCE STATEMENT: Compound 13, a reported selective GPR4 antagonist, has been widely used to assess the therapeutic potential of GPR4, a pH-sensing receptor, for numerous indications. Its pH dependence and mechanism of inhibition identified in this study clearly highlights the limitations of this chemotype for target validation.

Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention. DESIGN: Liver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention. RESULTS: In patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients. CONCLUSION: Our results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty acid ß-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in patients with NASH; however, the effect of ACC inhibition on liver fibrosis has not been reported. METHODS: A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, diethylnitrosamine. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. RESULTS: Inhibition of ACC reduced the activation of TGF-ß-stimulated HSCs, as measured by both α-SMA expression and collagen production. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. While the molecular mechanism by which inhibition of de novo lipogenesis blocks glycolysis and oxidative phosphorylation is unknown, we definitively show that HSCs require de novo lipogenesis for activation. Consistent with this direct antifibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat choline-deficient, high-fat diet model and in response to chronic diethylnitrosamine-induced liver injury (in the absence of hepatic lipid accumulation). CONCLUSIONS: In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the profibrogenic activity of HSCs. Thus, small molecule inhibitors of ACC may lessen fibrosis by reducing lipotoxicity in hepatocytes and by preventing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH. LAY SUMMARY: Hepatic fibrosis is the most important predictor of liver-related outcomes in patients with non-alcoholic steatohepatitis (NASH). Small molecule inhibitors of acetyl-CoA carboxylase (ACC) reduce hepatic fat content and markers of liver injury in patients with NASH. Herein, we report that inhibition of ACC and de novo lipogenesis also directly suppress the activation of hepatic stellate cells - the primary cell responsible for generating fibrotic scar in the liver - and thus fibrosis. These data provide further evidence for the use of ACC inhibitors to treat patients with NASH and advanced fibrosis.

HCV-Induced Epigenetic Changes Associated With Liver Cancer Risk Persist After Sustained Virologic Response.

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers. METHODS: We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. RESULTS: We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance. CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.

Advanced MRI of Liver Fibrosis and Treatment Response in a Rat Model of Nonalcoholic Steatohepatitis.

Background Liver biopsy is the reference standard to diagnose nonalcoholic steatohepatitis (NASH) but is invasive with potential complications. Purpose To evaluate molecular MRI with type 1 collagen-specific probe EP-3533 and allysine-targeted fibrogenesis probe Gd-Hyd, MR elastography, and native T1 to characterize fibrosis and to assess treatment response in a rat model of NASH. Materials and Methods MRI was performed prospectively (June-November 2018) in six groups of male Wistar rats (a) age- and (b) weight-matched animals received standard chow (n = 12 per group); (c) received choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) for 6 weeks or (d) 9 weeks (n = 8 per group); (e) were fed 6 weeks of CDAHFD and switched to standard chow for 3 weeks (n = 12); (f) were fed CDAHFD for 9 weeks with daily treatment of elafibranor beginning at week 6 (n = 14). Differences in imaging measurements and tissue analyses among groups were tested with one-way analysis of variance. The ability of each imaging measurement to stage fibrosis was quantified by using area under the receiver operating characteristic curve (AUC) with quantitative digital pathology (collagen proportionate area [CPA]) as reference standard. Optimal cutoff values for distinguishing advanced fibrosis were used to assess treatment response. Results AUC for distinguishing fibrotic (CPA >4.8%) from nonfibrotic (CPA ≤4.8%) livers was 0.95 (95% confidence interval [CI]: 0.91, 1.00) for EP-3533, followed by native T1, Gd-Hyd, and MR elastography with AUCs of 0.90 (95% CI: 0.83, 0.98), 0.84 (95% CI: 0.74, 0.95), and 0.65 (95% CI: 0.51, 0.79), respectively. AUCs for discriminating advanced fibrosis (CPA >10.3%) were 0.86 (95% CI: 0.76, 0.97), 0.96 (95% CI: 0.90, 1.01), 0.84 (95% CI: 0.70, 0.98), and 0.74 (95% CI: 0.63, 0.86) for EP-3533, Gd-Hyd, MR elastography, and native T1, respectively. Gd-Hyd MRI had the highest accuracy (24 of 26, 92%; 95% CI: 75%, 99%) in identifying responders and nonresponders in the treated groups compared with MR elastography (23 of 26, 88%; 95% CI: 70%, 98%), EP-3533 (20 of 26, 77%; 95% CI: 56%, 91%), and native T1 (14 of 26, 54%; 95% CI: 33%, 73%). Conclusion Collagen-targeted molecular MRI most accurately detected early onset of fibrosis, whereas the fibrogenesis probe Gd-Hyd proved most accurate for detecting treatment response. © RSNA, 2020 Online supplemental material is available for this article.

Serum Angiopoietin-2 Predicts Mortality and Kidney Outcomes in Decompensated Cirrhosis.

Acute kidney injury in decompensated cirrhosis has limited therapeutic options, and novel mechanistic targets are urgently needed. Angiopoietin-2 is a context-specific antagonist of Tie2, a receptor that signals vascular quiescence. Considering the prominence of vascular destabilization in decompensated cirrhosis, we evaluated Angiopoietin-2 to predict clinical outcomes. Serum Angiopoietin-2 was measured serially in a prospective cohort of hospitalized patients with decompensated cirrhosis and acute kidney injury. Clinical characteristics and outcomes were examined over a 90-day period and analyzed according to Angiopoietin-2 levels. Primary outcome was 90-day mortality. Our study included 191 inpatients (median Angiopoietin-2 level 18.2 [interquartile range 11.8, 26.5] ng/mL). Median Model for End-Stage Liver Disease (MELD) score was 23 [17, 30] and 90-day mortality was 41%. Increased Angiopoietin-2 levels were associated with increased mortality (died 21.9 [13.9, 30.3] ng/mL vs. alive 15.2 [9.8, 23.0] ng/mL; P < 0.001), higher Acute Kidney Injury Network stage (stage I 13.4 [9.8, 20.1] ng/mL vs. stage II 20.0 [14.1, 26.2] ng/mL vs. stage III 21.9 [13.0, 29.5] ng/mL; P = 0.002), and need for renal replacement therapy (16.5 [11.3, 23.6] ng/mL vs. 25.1 [13.3, 30.3] ng/mL; P = 0.005). The association between Angiopoietin-2 and mortality was significant in unadjusted and adjusted Cox regression models (P ≤ 0.001 for all models), and improved discrimination for mortality when added to MELD score (integrated discrimination increment 0.067; P = 0.001). Conclusion: Angiopoietin-2 was associated with mortality and other clinically relevant outcomes in a cohort of patients with decompensated cirrhosis with acute kidney injury. Further experimental study of Angiopoietin/Tie2 signaling is warranted to explore its potential mechanistic and therapeutic role in this population.

The farnesoid X receptor agonist EDP-305 reduces interstitial renal fibrosis in a mouse model of unilateral ureteral obstruction.

Farnesoid X receptor (FXR) is a nuclear receptor that has emerged as a key regulator in the maintenance of hepatic steatosis, inflammation, and fibrosis. However, the role of FXR in renal fibrosis remains to be established. Here, we investigate the effects of the FXR agonist EDP-305 in a mouse model of tubulointerstitial fibrosis via unilateral ureteral obstruction (UUO). Male C57Bl/6 mice received a UUO on their left kidney. On postoperative d 4, mice received daily treatment by oral gavage with either vehicle control (0.5% methylcellulose) or 10 or 30 mg/kg EDP-305. All animals were euthanized on postoperative d 12. EDP-305 dose-dependently decreased macrophage infiltration as measured by the F4/80 staining area and proinflammatory cytokine gene expression. EDP-305 also dose-dependently reduced interstitial fibrosis as assessed by morphometric quantification of the collagen proportional area and kidney hydroxyproline levels. Finally, yes-associated protein (YAP) activation, a major driver of fibrosis, increased after UUO injury and was diminished by EDP-305 treatment. Consistently, EDP-305 decreased TGF-ß1-induced YAP nuclear localization in human kidney 2 cells by increasing inhibitory YAP phosphorylation. YAP inhibition may be a novel antifibrotic mechanism of FXR agonism, and EDP-305 could be used to treat renal fibrosis.-Li, S., Ghoshal, S., Sojoodi, M., Arora, G., Masia, R., Erstad, D. J., Ferriera, D. S., Li, Y., Wang, G., Lanuti, M., Caravan, P., Or, Y. S., Jiang, L.-J., Tanabe, K. K., Fuchs, B. C. The farnesoid X receptor agonist EDP-305 reduces interstitial renal fibrosis in a mouse model of unilateral ureteral obstruction.

Molecular Magnetic Resonance Imaging Using a Redox-Active Iron Complex.

We introduce a redox-active iron complex, Fe-PyC3A, as a biochemically responsive MRI contrast agent. Switching between Fe3+-PyC3A and Fe2+-PyC3A yields a full order of magnitude relaxivity change that is field-independent between 1.4 and 11.7 T. The oxidation of Fe2+-PyC3A to Fe3+-PyC3A by hydrogen peroxide is very rapid, and we capitalized on this behavior for the molecular imaging of acute inflammation, which is characterized by elevated levels of reactive oxygen species.  Injection of Fe2+-PyC3A generates strong, selective contrast enhancement of inflamed pancreatic tissue in a mouse model (caerulein/LPS model). No significant signal enhancement is observed in normal pancreatic tissue (saline-treated mice). Importantly, signal enhancement of the inflamed pancreas correlates strongly and significantly with ex vivo quantitation of the pro-inflammatory biomarker myeloperoxidase. This is the first example of using metal ion redox for the MR imaging of pathologic change in vivo. Redox-active Fe3+/2+ complexes represent a new design paradigm for biochemically responsive MRI contrast agents.

68Ga-NODAGA-Indole: An Allysine-Reactive Positron Emission Tomography Probe for Molecular Imaging of Pulmonary Fibrogenesis.

Oxidized collagen, wherein lysine residues are converted to the aldehyde allysine, is a universal feature of fibrogenesis, i.e. actively progressive fibrosis. Here we report the small molecule, allysine-binding positron emission tomography probe, 68Ga-NODAGA-indole, that can noninvasively detect and quantify pulmonary fibrogenesis. We demonstrate that the uptake of 68Ga-NODAGA-indole in actively fibrotic lungs is 7-fold higher than in control groups and that uptake is linearly correlated ( R2 = 0.98) with the concentration of lung allysine.

Tyrosine kinase SYK is a potential therapeutic target for liver fibrosis.

Spleen tyrosine kinase (SYK) plays a critical role in immune cell signaling pathways and has been reported as a biomarker for human hepatocellular carcinoma (HCC). We sought to investigate the mechanism by which SYK promotes liver fibrosis and to evaluate SYK as a therapeutic target for liver fibrosis. We evaluated the cellular localization of SYK and the association between SYK expression and liver fibrogenesis in normal, hepatitis B virus (HBV)-infected, hepatitis C virus (HCV)-infected and non-alcoholic steatohepatitis (NASH) liver tissue (n=36, 127, 22 and 30, respectively). A polymerase chain reaction (PCR) array was used to detect the changes in transcription factor (TF) expression in hepatic stellate cells (HSCs) with SYK knockdown. The effects of SYK antagonism on liver fibrogenesis were studied in LX-2 cells, TWNT-4 cells, primary human HSCs, and three progressive fibrosis/cirrhosis animal models, including a CCL4 mouse model, and diethylnitrosamine (DEN) and bile duct ligation (BDL) rat models. We found that SYK protein in HSCs and hepatocytes correlated positively with liver fibrosis stage in human liver tissue. HBV or HCV infection significantly increased SYK and cytokine expression in hepatocytes. Increasing cytokine production further induced SYK expression and fibrosis-related gene transcription in HSCs. Up-regulated SYK in HSCs promoted HSC activation by increasing the expression of specific TFs related to activation of HSCs. SYK antagonism effectively suppressed liver fibrosis via inhibition of HSC activation, and decreased obstructive jaundice and reduced HCC development in animal models. Conclusion: SYK promotes liver fibrosis via activation of HSCs and is an attractive potential therapeutic target for liver fibrosis and prevention of HCC development. (Hepatology 2018).

Molecular signatures in hepatocellular carcinoma: A step toward rationally designed cancer therapy.

Molecular characterization of hepatocellular carcinoma (HCC) has greatly improved our understanding of disease pathogenesis. Mutational analysis, RNA and microRNA expression profiling, and epigenetic characterization have revealed common aberrations in oncogenes and tumor suppressors that correlate with disease biology and serve as a guide for the rational design of targeted therapies. These approaches have also led to the discovery of novel targets, including mutations in isocitrate dehydrogenase and chromatin remodeling enzymes. With the advent of immunotherapy, RNA expression profiling of the tumor microenvironment has identified a subset of HCC with high lymphocyte infiltration that may benefit from checkpoint inhibitor therapy. Molecular signatures thus capture the biology of a tumor, providing a supplement to current staging schema, which are based on tumor size and number, for more accurate prognostication of recurrence risk and survival. Molecular signatures may also be used to guide interventional therapy by defining those most suitable for transplantation or locoregional therapy rather than surgical resection. Finally, a multiomics approach involves the aggregation and analysis of multiple signatures for a more comprehensive characterization of pathogenic mechanisms. This broader approach attempts to address issues with signaling pathway cross-talk and redundancy, which have greatly limited the potential value of targeted therapies to date. Cancer 2018. © 2018 American Cancer Society.

CM-101: Type I Collagen-targeted MR Imaging Probe for Detection of Liver Fibrosis.

Purpose To evaluate the biodistribution, metabolism, and pharmacokinetics of a new type I collagen-targeted magnetic resonance (MR) probe, CM-101, and to assess its ability to help quantify liver fibrosis in animal models. Materials and Methods Biodistribution, pharmacokinetics, and stability of CM-101 in rats were measured with mass spectrometry. Bile duct-ligated (BDL) and sham-treated rats were imaged 19 days after the procedure by using a 1.5-T clinical MR imaging unit. Mice were treated with carbon tetrachloride (CCl4) or with vehicle two times a week for 10 weeks and were imaged with a 7.0-T preclinical MR imaging unit at baseline and 1 week after the last CCl4 treatment. Animals were imaged before and after injection of 10 µmol/kg CM-101. Change in contrast-to-noise ratio (ΔCNR) between liver and muscle tissue after CM-101 injection was used to quantify liver fibrosis. Liver tissue was analyzed for Sirius Red staining and hydroxyproline content. The institutional subcommittee for research animal care approved all in vivo procedures. Results CM-101 demonstrated rapid blood clearance (half-life = 6.8 minutes ± 2.4) and predominately renal elimination in rats. Biodistribution showed low tissue gadolinium levels at 24 hours (<3.9% injected dose [ID]/g ± 0.6) and 10-fold lower levels at 14 days (<0.33% ID/g ± 12) after CM-101 injection with negligible accumulation in bone (0.07% ID/g ± 0.02 and 0.010% ID/g ± 0.004 at 1 and 14 days, respectively). ΔCNR was significantly (P < .001) higher in BDL rats (13.6 ± 3.2) than in sham-treated rats (5.7 ± 4.2) and in the CCl4-treated mice (18.3 ± 6.5) compared with baseline values (5.2 ± 1.0). Conclusion CM-101 demonstrated fast blood clearance and whole-body elimination, negligible accumulation of gadolinium in bone or tissue, and robust detection of fibrosis in rat BDL and mouse CCl4 models of liver fibrosis. © RSNA, 2017 Online supplemental material is available for this article.

Combined magnetic resonance elastography and collagen molecular magnetic resonance imaging accurately stage liver fibrosis in a rat model.

Hepatic fibrosis is associated with an overproduction of matrix proteins and a pathological increase of liver stiffness. Noninvasive magnetic resonance (MR) quantification of matrix can be assessed with a collagen-binding molecular MR probe and stiffness by MR elastography, complementary techniques. This study used both imaging techniques to more accurately stage hepatic fibrosis in a rat model. Thirty rats with varying levels of diethylnitrosamine-induced liver fibrosis were imaged before and 45 minutes after injection of collagen-specific probe EP-3533. MR elastography was performed in the same imaging session. Changes in liver relaxation rate post-EP-3533 and liver stiffness were compared to the collagen proportional area determined by histology and to Ishak scoring using receiver operating characteristic analysis. Collagen imaging was most sensitive to early fibrosis, while elastography was more sensitive to advanced fibrosis. This complementary feature enabled the formulation of a composite model using multivariate analysis of variance. This model incorporated the discriminating advantages of both MR techniques, resulting in more accurate staging throughout fibrotic progression. CONCLUSION: Collagen molecular MR imaging is complementary to MR elastography, and combining the two techniques in a single exam leads to increased diagnostic accuracy for all stages of fibrosis. (Hepatology 2017;65:1015-1025).

Cell type-specific pharmacological kinase inhibition for cancer chemoprevention.

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention.

Molecular subclasses of hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition.

A recent gene expression classification of hepatocellular carcinoma (HCC) includes a poor survival subclass termed S2 representing about one-third of all HCC in clinical series. S2 cells express E-cadherin and c-myc and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between S2 and non-S2 HCC, this study investigated whether molecular subclasses of HCC predict sensitivity to FGFR inhibition. S2 cell lines were significantly more sensitive (p < 0.001) to the FGFR inhibitors BGJ398 and AZD4547. BGJ398 decreased MAPK signaling in S2 but not in non-S2 cell lines. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 44% or more in all five S2 cell lines (p < 0.05 for each cell line), a significantly greater decrease than seen with knockdown of FGFR1-3 with siRNA transfection. FGFR4 knockdown decreased MAPK signaling in S2 cell lines, but little effect was seen with knockdown of FGFR1-3. In conclusion, the S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4-MAPK signaling plays an important role in driving proliferation of a molecular subclass of HCC. This classification system may help to identify those patients who are most likely to benefit from inhibition of this pathway.

Metformin prevents hepatocellular carcinoma development by suppressing hepatic progenitor cell activation in a rat model of cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC)-associated mortality is increasing at an alarming rate, and there is a readily identifiable cohort of at-risk patients with cirrhosis, viral hepatitis, nonalcoholic fatty liver disease, and diabetes. These patients are candidates for chemoprevention. Metformin is an attractive agent for chemoprevention because it is inexpensive, has a favorable safety profile, and is well tolerated over long time periods. METHODS: The authors studied the efficacy of metformin as a prevention agent in a clinically relevant rat model of HCC, in which tumors develop in the setting of chronic inflammation and cirrhosis. Repeated injections of diethylnitrosamine were used to induce sequential cirrhosis and HCC, and metformin was administered at the first signs of either fibrosis or cirrhosis. RESULTS: Prolonged metformin exposure was safe and was associated with decreases in fibrotic and inflammatory markers, especially when administered early at the first signs of fibrosis. In addition, early metformin treatment led to a 44% decrease in HCC incidence, whereas tumor burden was unchanged when metformin was administered at the first signs of cirrhosis. It is noteworthy that activation of the hepatic progenitor/stem cell compartment was first observed at the onset of cirrhosis; therefore, only early metformin treatment suppressed receptor for advanced glycation end products and inhibited the activation of hepatic progenitor cells. CONCLUSIONS: The current results are the first to demonstrate an effect on progenitor/stem cells in the setting of chemoprevention and provide further rationale to explore metformin as an early intervention in clinical trials of patients with chronic liver disease at high risk for HCC.

Impact of EGF, IL28B, and PNPLA3 polymorphisms on the outcome of allograft hepatitis C: a multicenter study.

Hepatitis C virus (HCV) infection is accelerated following liver transplantation (LT). Single nucleotide polymorphisms (SNPs) near the epidermal growth factor (EGF) (rs4444903), IL28B (rs12979860), and PNPLA3 (rs738409) loci are associated with treatment response, fibrosis, and hepatocellular carcinoma in non-transplant hepatitis C, but allograft population data are limited. We sought to determine the role of these SNPs in 264 patients with HCV who underwent LT between 1990 and 2008. Genotypes were determined from donor wedge/allograft biopsies and recipient explants. Cox proportional hazards model was used to assess time to cirrhosis, liver-related death, and retransplantation, adjusting for donor age and sustained virological response (SVR). Over a median follow-up of 6.3 yr, a trend toward increased progression to graft cirrhosis was observed among recipients of an EGF non-AA vs. AA donor liver (adjusted HR 2.01; 95% CI 0.93-4.34; p = 0.08). No other genotypes predicted cirrhosis development or graft survival. The CC IL28B variant in both recipients and donors was associated with increased rate of SVR (R-CC/D-CC 8/12[67%], R-non-CC/D-CC or R-CC/D-non-CC 23/52[44%], R-non-CC/D-non-CC 12/45[27%], p linear trend = 0.009). Recipient EGF, IL28B, and PNPLA3, and donor IL28B and PNPLA3 genotypes do not predict adverse outcomes in HCV LT recipients. A potential association exists between donor EGF genotype and cirrhosis.

3D molecular MR imaging of liver fibrosis and response to rapamycin therapy in a bile duct ligation rat model.

BACKGROUND & AIMS: Liver biopsy, the gold standard for assessing liver fibrosis, suffers from limitations due to sampling error and invasiveness. There is therefore a critical need for methods to non-invasively quantify fibrosis throughout the entire liver. The goal of this study was to use molecular Magnetic Resonance Imaging (MRI) of Type I collagen to non-invasively image liver fibrosis and assess response to rapamycin therapy. METHODS: Liver fibrosis was induced in rats by bile duct ligation (BDL). MRI was performed 4, 10, or 18 days following BDL. Some BDL rats were treated daily with rapamycin starting on day 4 and imaged on day 18. A three-dimensional (3D) inversion recovery MRI sequence was used to quantify the change in liver longitudinal relaxation rate (ΔR1) induced by the collagen-targeted probe EP-3533. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for Sirius Red staining and hydroxyproline content. RESULTS: ΔR1 increased significantly with time following BDL compared to controls in agreement with ex vivo measures of increasing fibrosis. Receiver operating characteristic curve analysis demonstrated the ability of ΔR1 to detect liver fibrosis and distinguish intermediate and late stages of fibrosis. EP-3533 MRI correctly characterized the response to rapamycin in 11 out of 12 treated rats compared to the standard of collagen proportional area (CPA). 3D MRI enabled characterization of disease heterogeneity throughout the whole liver. CONCLUSIONS: EP-3533 allowed for staging of liver fibrosis, assessment of response to rapamycin therapy, and demonstrated the ability to detect heterogeneity in liver fibrosis.