Molecular imaging of oxidized collagen quantifies pulmonary and hepatic fibrogenesis

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Abstract

Fibrosis results from the dysregulation of tissue repair mechanisms affecting major organ systems, leading to chronic extracellular matrix buildup, and progressive, often fatal, organ failure. Current diagnosis relies on invasive biopsies. Noninvasive methods today cannot distinguish actively progressive fibrogenesis from stable scar, and thus are insensitive for monitoring disease activity or therapeutic responses. Collagen oxidation is a universal signature of active fibrogenesis that precedes collagen crosslinking. Biochemically targeting oxidized lysine residues formed by the action of lysyl oxidase on collagen with a small-molecule gadolinium chelate enables targeted molecular magnetic resonance imaging. This noninvasive direct biochemical elucidation of the fibrotic microenvironment specifically and robustly detected and staged pulmonary and hepatic fibrosis progression, and monitored therapeutic response in animal models. Furthermore, this paradigm is translatable and generally applicable to diverse fibroproliferative disorders.

Authors

Howard H. Chen, Philip A. Waghorn, Lan Wei, Luis F. Tapias, Daniel T. Schühle, Nicholas J. Rotile, Chloe M. Jones, Richard J. Looby, Gaofeng Zhao, Justin M. Elliott, Clemens K. Probst, Mari Mino-Kenudson, Gregory Y. Lauwers, Andrew M. Tager, Kenneth K. Tanabe, Michael Lanuti, Bryan C. Fuchs, Peter Caravan

E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9 expression in the liver

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Abstract

Cholesterol accumulation in the liver is an early event in nonalcoholic fatty liver disease (NAFLD). Here, we demonstrate that E2F1 plays a crucial role in maintaining cellular cholesterol homeostasis by regulating cholesterol uptake via proprotein convertase subtilisin/kexin 9 (PCSK9), an enzyme that promotes low-density lipoprotein receptor (LDLR) degradation upon activation. E2f1^–/– mice display reduced total plasma cholesterol levels and increased cholesterol content in the liver. In this study, we show that E2f1 deletion in cellular and mouse models leads to a marked decrease in Pcsk9 expression and an increase in LDLR expression. In addition to the upregulation of LDLR, we report that E2f1^–/– hepatocytes exhibit increased LDL uptake. ChIP-Seq and PCSK9 promoter reporter experiments confirmed that E2F1 binds to and transactivates the PCSK9 promoter. Interestingly, E2f1^–/– mice fed a high-cholesterol diet (HCD) display a fatty liver phenotype and liver fibrosis, which is reversed by reexpression of PCSK9 in the liver. Collectively, these data indicate that E2F1 regulates cholesterol uptake and that the loss of E2F1 leads to abnormal cholesterol accumulation in the liver and the development of fibrosis in response to an HCD.

Authors

Qiuwen Lai, Albert Giralt, Cédric Le May, Lianjun Zhang, Bertrand Cariou, Pierre-Damien Denechaud, Lluis Fajas

Bile acids initiate cholestatic liver injury by triggering a hepatocyte-specific inflammatory response

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Abstract

Mechanisms of bile acid–induced (BA-induced) liver injury in cholestasis are controversial, limiting development of new therapies. We examined how BAs initiate liver injury using isolated liver cells from humans and mice and in-vivo mouse models. At pathophysiologic concentrations, BAs induced proinflammatory cytokine expression in mouse and human hepatocytes, but not in nonparenchymal cells or cholangiocytes. These hepatocyte-specific cytokines stimulated neutrophil chemotaxis. Inflammatory injury was mitigated in Ccl2^–/– mice treated with BA or after bile duct ligation, where less hepatic infiltration of neutrophils was detected. Neutrophils in periportal areas of livers from cholestatic patients also correlated with elevations in their serum aminotransferases. This liver-specific inflammatory response required BA entry into hepatocytes via basolateral transporter Ntcp. Pathophysiologic levels of BAs induced markers of ER stress and mitochondrial damage in mouse hepatocytes. Chemokine induction by BAs was reduced in hepatocytes from Tlr9^–/– mice, while liver injury was diminished both in conventional and hepatocyte-specific Tlr9^–/– mice, confirming a role for Tlr9 in BA-induced liver injury. These findings reveal potentially novel mechanisms whereby BAs elicit a hepatocyte-specific cytokine-induced inflammatory liver injury that involves innate immunity and point to likely novel pathways for treating cholestatic liver disease.

Authors

Shi-Ying Cai, Xinshou Ouyang, Yonglin Chen, Carol J. Soroka, Juxian Wang, Albert Mennone, Yucheng Wang, Wajahat Z. Mehal, Dhanpat Jain, James L. Boyer

Liver X receptor α mediates hepatic triglyceride accumulation through upregulation of G0/G1 Switch Gene 2 expression

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Abstract

Liver X receptors (LXRs) are transcription factors essential for cholesterol homeostasis and lipogenesis. LXRα has been implicated in regulating hepatic triglyceride (TG) accumulation upon both influx of adipose-derived fatty acids (FAs) during fasting and stimulation of de novo FA synthesis by chemical agonism of LXR. However, whether or not a convergent mechanism is employed to drive deposition of FAs from these 2 different sources in TGs is undetermined. Here, we report that the G0/G1 Switch Gene 2 (G0S2), a selective inhibitor of intracellular TG hydrolysis/lipolysis, is a direct target gene of LXRα. Transcriptional activation is conferred by LXRα binding to a direct repeat 4 (DR4) motif in the G0S2 promoter. While LXRα^–/– mice exhibited decreased hepatic G0S2 expression, adenoviral expression of G0S2 was sufficient to restore fasting-induced TG storage and glycogen depletion in the liver of these mice. In response to LXR agonist T0901317, G0S2 ablation prevented hepatic steatosis and hypertriglyceridemia without affecting the beneficial effects on HDL. Thus, the LXRα-G0S2 axis plays a distinct role in regulating hepatic TG during both fasting and pharmacological activation of LXR.

Authors

Bradlee L. Heckmann, Xiaodong Zhang, Alicia M. Saarinen, Gabriele Schoiswohl, Erin E. Kershaw, Rudolf Zechner, Jun Liu

IRF5 governs liver macrophage activation that promotes hepatic fibrosis in mice and humans

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Abstract

Hepatic fibrosis arises from inflammation in the liver initiated by resident macrophage activation and massive leukocyte accumulation. Hepatic macrophages hold a central position in maintaining homeostasis in the liver and in the pathogenesis of acute and chronic liver injury linked to fibrogenesis. Interferon regulatory factor 5 (IRF5) has recently emerged as an important proinflammatory transcription factor involved in macrophage activation under acute and chronic inflammation. Here, we revealed that IRF5 is significantly induced in liver macrophages from human subjects developing liver fibrosis from nonalcoholic fatty liver disease or hepatitis C virus infection. Furthermore, IRF5 expression positively correlated with clinical markers of liver damage, such as plasma transaminase and bilirubin levels. Interestingly, mice lacking IRF5 in myeloid cells (MKO) were protected from hepatic fibrosis induced by metabolic or toxic stresses. Transcriptional reprogramming of macrophages lacking IRF5 was characterized by immunosuppressive and antiapoptotic properties. Consequently, IRF5 MKO mice respond to hepatocellular stress by promoting hepatocyte survival, leading to complete protection from hepatic fibrogenesis. Our findings reveal a regulatory network, governed by IRF5, that mediates hepatocyte death and liver fibrosis in mice and humans. Therefore, modulating IRF5 function may be an attractive approach to experimental therapeutics in fibroinflammatory liver disease.

Authors

Fawaz Alzaid, Floriane Lagadec, Miguel Albuquerque, Raphaëlle Ballaire, Lucie Orliaguet, Isabelle Hainault, Corinne Blugeon, Sophie Lemoine, Agnès Lehuen, David G. Saliba, Irina A. Udalova, Valérie Paradis, Fabienne Foufelle, Nicolas Venteclef

Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis

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Abstract

A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies. The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase, P < 0.01) and a lipidomics signature similar to NASH biopsies. Hepatocyte glucose output increased with decreased insulin sensitivity. These changes were accompanied by increased inflammatory analyte secretion (e.g., IL-6, IL-8, alanine aminotransferase). Fibrogenic activation markers increased with lipotoxic conditions, including secreted TGF-β (>5-fold increase, P < 0.05), extracellular matrix gene expression, and HSC activation. Significant pathway correlation existed between this in vitro model and human biopsies. Consistent with clinical trial data, 0.5 μM obeticholic acid in this model promoted a healthy lipidomic signature, reduced inflammatory and fibrotic secreted factors, but also increased ApoB secretion, suggesting a potential adverse effect on lipoprotein metabolism. Lipotoxic stress activates similar biological signatures observed in NASH patients in this system, which may be relevant for interrogating novel therapeutic approaches to treat NASH.

Authors

Ryan E. Feaver, Banumathi K. Cole, Mark J. Lawson, Stephen A. Hoang, Svetlana Marukian, Brett R. Blackman, Robert A. Figler, Arun J. Sanyal, Brian R. Wamhoff, Ajit Dash

Hepatoblastoma modeling in mice places Nrf2 within a cancer field established by mutant β-catenin

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Abstract

Aberrant wnt/β-catenin signaling and amplification/overexpression of Myc are associated with hepatoblastoma (HB), the most prevalent type of childhood liver cancer. To address their roles in the pathogenesis of HB, we generated mice in which Myc and mutant β-catenin were targeted to immature cells of the developing mouse liver. Perinatal coexpression of both genes promoted the preferential development of HBs over other tumor types in neonatal mice, all of which bore striking resemblance to their human counterparts. Integrated analysis indicated that tumors emerged as a consequence of Myc-driven alterations in hepatoblast fate in a background of pan-hepatic injury, inflammation, and nuclear factor (erythroid-derived 2)-like 2/Nrf2-dependent antioxidant signaling, which was specifically associated with expression of mutant β-catenin but not Myc. Immunoprofiling of human HBs confirmed that approximately 50% of tumors demonstrated aberrant activation of either Myc or Nfe2l2/Nrf2, while knockdown of Nrf2 in a cell line–derived from a human HB with NFE2L2 gene amplification reduced tumor cell growth and viability. Taken together, these data indicate that β-catenin creates a protumorigenic hepatic environment in part by indirectly activating Nrf2 and implicate oxidative stress as a possible driving force for a subset of β-catenin–driven liver tumors in children.

Authors

Sarah A. Comerford, Elizabeth A. Hinnant, Yidong Chen, Hima Bansal, Shawn Klapproth, Dinesh Rakheja, Milton J. Finegold, Dolores Lopez-Terrada, Kathryn A. O’Donnell, Gail E. Tomlinson, Robert E. Hammer

Quantum coherence spectroscopy to measure dietary fat retention in the liver

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Abstract

The prevalence of fatty liver reaches alarming proportions. Fatty liver increases the risk for insulin resistance, cardiovascular disease, and nonalcoholic steatohepatitis (NASH). Although extensively studied in a preclinical setting, the lack of noninvasive methodologies hampers our understanding of which pathways promote hepatic fat accumulation in humans. Dietary fat retention is one of the pathways that may lead to fatty liver. The low (1.1%) natural abundance (NA) of carbon-13 (¹³C) allows use of ¹³C-enriched lipids for in vivo MR studies. Successful implementation of such methodology, however, is challenging due to low sensitivity of ¹³C-magnetic resonance spectroscopy (¹³C-MRS). Here, we investigated the use of 1-dimensional gradient enhanced heteronuclear single quantum coherence (ge-HSQC) spectroscopy for the in vivo detection of hepatic ¹H-[¹³C]-lipid signals after a single high-fat meal with ¹³C-labeled fatty acids in 5 lean and 6 obese subjects. Postprandial retention of orally administered ¹³C-labeled fatty acids was significant (P < 0.01). Approximately 1.5% of the tracer was retained in the liver after 6 hours, and retention was similar in both groups (P = 0.92). Thus, a substantial part of the liver fat can originate directly from storage of meal-derived fat. The ge-HSQC can be used to noninvasively reveal the contribution of dietary fat to the development of hepatic steatosis over time.

Authors

Lucas Lindeboom, Robin A. de Graaf, Christine I. Nabuurs, Petronella A. van Ewijk, Matthijs K.C. Hesselink, Joachim E. Wildberger, Patrick Schrauwen, Vera B. Schrauwen-Hinderling

Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis

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Abstract

Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB₁R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB₁R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB₁R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl₄ or bile duct ligation, the hybrid CB₁R/iNOS antagonist surpassed the antifibrotic efficacy of the CB₁R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB₁R occupancy in the CNS. The hybrid inhibitor also targeted CB₁R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1^–/– but not in nos2^–/– mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB₁R/iNOS antagonists have therapeutic potential in liver fibrosis.

Authors

Resat Cinar, Malliga R. Iyer, Ziyi Liu, Zongxian Cao, Tony Jourdan, Katalin Erdelyi, Grzegorz Godlewski, Gergő Szanda, Jie Liu, Joshua K. Park, Bani Mukhopadhyay, Avi Z. Rosenberg, Jeih-San Lieow, Robin G. Lorenz, Pal Pacher, Robert B. Innis, George Kunos

CRIg-expressing peritoneal macrophages are associated with disease severity in patients with cirrhosis and ascites

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Abstract

Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesizing that innate immune dysfunction contributes to susceptibility to infection, we assessed ascitic fluid macrophage phenotype and function. The expression of complement receptor of the immunoglobulin superfamily (CRIg) and CCR2 defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. The proportion of CRIg^hi macrophages differed between patients and in the same patient over time, and a high proportion of CRIg^hi macrophages was associated with reduced disease severity (model for end-stage liver disease) score. As compared with CRIg^lo macrophages, CRIg^hi macrophages were highly phagocytic and displayed enhanced antimicrobial effector activity. Transcriptional profiling by RNA sequencing and comparison with human macrophage and murine peritoneal macrophage expression signatures highlighted similarities among CRIg^hi cells, human macrophages, and mouse F4/80^hi resident peritoneal macrophages and among CRIg^lo macrophages, human monocytes, and mouse F4/80^lo monocyte-derived peritoneal macrophages. These data suggest that CRIg^hi and CRIg^lo macrophages may represent a tissue-resident population and a monocyte-derived population, respectively. In conclusion, ascites fluid macrophage subset distribution and phagocytic capacity is highly variable among patients with chronic liver disease. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities in cirrhotic patients.

Authors

Katharine M. Irvine, Xuan Banh, Victoria L. Gadd, Kyle K. Wojcik, Juliana K. Ariffin, Sara Jose, Samuel Lukowski, Gregory J. Baillie, Matthew J. Sweet, Elizabeth E. Powell