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Liver cell plasticity Prof Anna Mae Diehl Duke University, Durham, United States

The injured liver’s ability to withstand noxious insults and to regenerate while maintaining its vital functions distinguish it from other adult organs. This unique resiliency reflects a beautifully scripted collaborative response that derives from the inherent plasticity of adult liver cells. This cellular “nimbleness” enables some surviving liver cells to become more proliferative to regenerate healthy liver tissue. At that same time, the organ assumes new repair-related activities while compensating for the loss of cells that were previously performing vital liver-specific duties. Liver diseases (cirrhosis and liver failure) ensue when mechanisms that regulate and coordinate these critical adaptive responses become dysregulated. Thus, defining tractable mechanisms that control liver cell plasticity will facilitate prevention/reversal of these diseases. Our research indicates that mechanisms that control cell fates during liver development also orchestrate changes in liver cell fates that are necessary for adult livers to regenerate. For example, studies of adult hepatocytes (Heps) and hepatic stellate cells (HSCs) indicate that both cell types actively repress their proliferative phenotypes in healthy livers. Our findings indicate that injury-related cytokines can rapidly release this repression by differentially mobilizing/silencing RNA binding proteins that regulate the stability, splicing, and translation of pre-existing mRNAs that encode proteins that control the activity of transcriptional regulators of epithelial-mesenchymal transitions and liver growth (e.g., YAP1). The injury-adapted Heps and HSCs, in turn, generate signals that modulate phenotypic changes in other types of liver-resident and infiltrating cells and these cells then further modulate Hep and HSC fates in order to orchestrate rapid remodelling of damaged hepatic epithelia into healthy parenchyma. Either under- and over-activation of these mechanisms derails regeneration and drives progressive loss of functional hepatocytes and excessive accumulation of fibro genic cells. Organ fibrosis and failure eventually result, recapitulating the typical dismal outcomes of injury in other vital, but less regeneratively-gifted, organs.

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101

Cirrhosis of the liver: A vascular disease

Prof Jaime Bosch Hepatology, Department for Biomedical Research, Inselspital, University of Bern, Switzerland Cirrhosis of the liver represents the final step of chronic liver disease of different etiologies (alcohol abuse, chronic viral hepatitis, non-alcoholic steatohepatitis, and autoimmune hepatitis, among others). It is characterized by the formation of regenerating nodules surrounded by fibrous septa. Parenchymal extinction due to vascular occlusion further contributes to macroscopic liver remodeling. A closer look to the cirrhotic liver discloses marked vascular distortion, with irregular sinusoids, increased number of arteries per surface area (reflecting parenchymal extinction), and abnormally spaced veins. The liver stiffness is much increased. Vascular casts reveal dramatic changes at the liver microcirculation. On transmission EM, the liver sinusoidal cells (LSEC) appear capillarized by the development of a basement membrane and loss fenestrations. Liver perfusion discloses a markedly increased perfusion pressure, increased sensitivity to vasoconstrictors, and markedly impaired endothelial dependent vasorelaxation (“endothelial dysfunction”) thus indicating the existence of an increased resistance to portal blood flow and increased hepatic vascular tone, which are responsible for the development of portal hypertension, the major clinical complication of cirrhosis. Liver cells change their phenotype, which is particularly prominent in the hepatic stellate cells (HSC) that become activated, loss vitamin A deposits, proliferate, become contractile and produced increased amounts of extracellular matrix leading to fibrogenesis; LSEC in turn become pro-angiogenic, pro-inflammatory, pro-thrombotic, and provasoconstrictive, and liver resident macrophages (Kupffer cells, KC) become activated and secrete increased amounts of pro-inflammatory citokines. Liver cells interact with each other by means of their secretome, and also through microvesicle release, especially from hepatocytes (Ho). Regression of cirrhosis is possible by suppressing the etiologic factor and by reversing the phenotypic changes of liver cells. Of special importance is acting on LSEC and HSC. This has been well illustrated by multiple experimental studies indicating enhanced cirrhosis regression by the administration of statins (simvastatin, atorvastatin), CGM activators/enhancers and of strong antioxydants. Acting on hepatocytes by means of FXR-agonists, PPAR blockers or caspase inhibitors has also been succesfull. Positive clinical data is only available for the use of statins and for the FXR agonist obeticholic acid. There are some data suggesting a beneficial effect of enoxaparin on liver fibrosis, by this is still controversial. Therefore, both from a pathological, physiological, molecular and therapeutic point of view there are enough similarities between the pathobiology of cirrhosis and that of the vascular wall in atherosclerosis as to indicate that cirrhosis should be considered as a vascular disease of the liver.

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102

Reinvigorating old holes in the ageing liver, and how to watch this happen

Assoc Prof Peter McCourt Dept. Medical Biology, University of Tromsø, Norway When we age, our liver undergoes a morphological transformation termed "defenestration" whereby liver sinusoidal endothelial cells (LSEC) lose fenestrations (pores ≤ 200 nm) that perforate the LSEC. These pores facilitate the 2-way transfer of biomolecules to and from the plasma and the underlying hepatocytes, and their loss has a significant impact on systemic ageing. Recent work, however, has shown that defenestration may be reversible, and promising new therapies may arise from this research. Nevertheless, the study of fenestrations suffers from a "tyranny of distance" namely the 250 nm optical diffraction limit of visible light. This limit has prevented us from using light microscopy to view fenestrations - until recently, these structures were only discernible with electron (EM) and atomic force (AFM) microscopy, but now they can be visualized with optical methods in fixed and living cells using structured illumination (SIM) and Stimulated Emission Depletion (STED) microscopy, and in fixed cells using single molecule localization microscopy (SMLM) techniques such as direct stochastic optical reconstruction microscopy (dSTORM). And while EM remains the "gold-standard" for morphology, and AFM allows label-free examination of fenestrations, SIM/STED and dSTORM permit imaging in multiple colour channels. These relatively new modalities will thus enable us to determine the components and structure of fenestrations, and (ultimately) how they are regulated. A combination of some (or all) of the above methods will likely be necessary in our search for therapies that can reverse age-related defenestration of LSEC in the elderly.

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103

Macrophages in liver diseases

Prof Frank Tacke Department of Hepatology & Gastroenterology, Charité University Medical Center, Berlin, Germany Macrophages represent a key innate immune cell component of the liver. They act as tissue response orchestrators that maintain homeostasis and coordinate rapid responses to hepatic injury. Thus, macrophages are essentially involved in the pathogenesis of acute and chronic liver diseases, including rapid inflammatory responses after injury, hepatic fibrosis progression or regression as well as transition from chronic inflammation to hepatocellular carcinoma (HCC). Two discoveries have revolutionized our understanding of liver macrophages: (a) The recognition of heterogeneous subsets such as liver-resident, self-sustaining Kupffer cells as opposed to monocyte-derived, infiltrating macrophages that rapidly accumulate in injured liver. (b) The notion of macrophage plasticity, which allows them to adapt their phenotype in response to local and systemic signals, thereby providing a large spectrum of functional responses. Recent findings revealed how hepatic macrophages can fulfill diverse and even opposing functions in homeostasis, disease progression and regression from injury. Understanding hepatic macrophage heterogeneity, up to a single cell level, and the underlying mechanisms may help to develop novel macrophage subset-targeted therapies for liver disease. The recent increased prevalence of dysmetabolic conditions such as obesity, dyslipidemia and type 2 diabetes concur to steadily growing numbers of patients suffering from nonalcoholic fatty liver disease (NAFLD) and its associated pathologies, namely fibrosis, cirrhosis and HCC. Consequently, NAFLD has become the most common soil for liver disease in industrialized countries, representing an immense clinical and economic burden notably through its potential progression from steatosis toward non-alcoholic steatohepatitis (NASH). Strategies to modulate macrophage activation, whether by preventing or favoring inflammatory or restorative macrophage populations, are currently explored in preclinical and clinical studies for liver disease treatment.

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TGF-β1 transcriptionally repressed cytoglobin expression and enhanced H2O2-induced oxidative DNA damage in activated HSCs. Misako Sato-Matsubara1, 2, Yoshinori Okina1, Tsutomu Matsubara3, Atsuko Daikoku1, Krista Rombouts4, Le Thi Thanh Thuy1, Hideki Fujii1, Kazuo Ikeda3, Katsutoshi Yoshizato2, Massimo Pinzani4, and Norifumi Kawada1 Departments of Hepatology1, Endowed Laboratory of Synthetic Biology2, Anatomy and Regenerative Biology3, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan, Regenerative Medicine and Fibrosis Group, Institute for Liver and Digestive Health, University College London, Royal Free Hospital, London, NW3 2PF, United Kingdom4

Introduction. Cytoglobin (CYGB) is a recently discovered respiratory protein with function as a radical scavenger. Although CYGB is expressed uniquely in hepatic stellate cells (HSCs) in the liver, molecular role of CYGB in human NASH remains uncharacterized. Aims. The goals of this study to reveal a regulatory mechanism of human CYGB gene promoter by TGF-β1/SMADs signal and physiological function of CYGB in human NASH fibrosis. Methods. Human NASH specimens, primary mouse, human HSCs and HSC line (HHSteCs) were used in this study. Results. 4-HNE and 8-OHdG, respective markers of oxidative damage for lipid peroxidation and DNA damage, showed increased number of positive cells in advanced fibrosis. In addition, the overexpression of CYGB suppressed TGF-β1/H2O2-induced intracellular ROS activity. Treatment of HHSteCs with 5 ng/mL TGF-β1 significantly suppressed CYGB mRNA (< 61%) and promoted αSMA mRNA (> 57%) expressions compared with untreated ones, whereas TGF-β1 failed to change CYGB expression in primary mouse HSCs. SiRNA knockdown system revealed that TGF-β1 reduced CYGB expression via SMAD2 and increased αSMA expression via SMAD3. A putative SP1/3 response element (+2) is present in transcriptional initiation site of human CYGB promoter, but absent in that of mouse Cygb promoter. TGF-β1 suppressed luciferase activity of human CYGB promoter (-2133/+73), which was abolished by mutating the SP1/3 response element. ChIP and IP analyses indicated that TGF-β1/Smad2 reduced CYGB transcription by recruiting SP3, but not SP1, to CYGB promoter in HHSteCs. Finally, CYGB expression was absent in pSMAD2+8-OHdG+ stromal cells of NASH with advanced fibrosis. Discussion. From these observations, we concluded that TGF-β enhanced ROS-induced oxidative DNA damage following downregulation of CYGB expression in HSCs of NASH patients. Our research provides new insights into the pathophysiology of human NASH fibrosis, which opens a novel approach for NASH therapy.

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Liver sinusoidal endothelial cell-specific knockdown of RUNX1 reduces hepatic inflammation and immune cell infiltration in mice models of non-alcoholic steatohepatitis

Savneet Kaur1, Dinesh M Tripathi1, Impreet Kaur1, Sumati Rohilla2, Abhishak Gupta1, Vikash Kumar3, Vegi Naidu4, Subham Banerjee4, Shiv K Sarin1. 1Institute of Liver and Biliary Sciences, New Delhi, India,2Gautam Buddha University, Greater Noida, India, 3National Institute of Immunology, New Delhi, India, 4National Institute of Pharmaceutical Education and Research, Guwahati, India.

Introduction: Runt-related transcription factor (RUNX1) is known to regulate both development and pathological angiogenesis. Aims: We dissected the role of RUNX1 in non-alcoholic steatohepatitis (NASH) by clinical, in vitro and in vivo studies. Methods: RUNX1 gene and protein expression was evaluated in liver biopsies in NASH patients and healthy subjects by RT-PCRs and by immunoflorescence (IF). In vitro, RUNX1 siRNAs were transfected in steatotic liver sinusoidal endothelial cells (LSECs) followed by analysis of their angiogenic gene expression and functions by RT-PCRs and matrigel assays. In vivo, RUNX1 was silenced in LSECs in methionine choline deficient (MCD) diet-induced NASH mice by using a novel nanodelivery system consisting of vegfr3-nanolipocarriers encapsulated RUNX1-siRNA. After characterization of the nanolipocarriers (both in vitro and in vivo), liver cells, liver infiltrating lymphocytes (LILs) and blood cells were analyzed to study expression of RUNX1 and other angiogenic/adhesion molecules by RT-PCRs and flow cytometery. MCD mice treated only with nanolipocarriers served as controls. Results: Liver RUNX1 gene and protein expression was enhanced in patients with NASH as compared to healthy subjects (p< 0.05). In dual IF staining of NASH liver, RUNX1 co-localized with vegfr3+ LSECs and CD45+ LILs. CD45+ blood cells also showed increased positivity of RUNX1 in patients. Both in vitro and in vivo knock-down of RUNX1 gene in steatotic LSECs (>50%) resulted in substantial downregulation of angiogenic, inflammatory and adhesion gene expression and also angiogenic properties. In vivo, no significant change in RUNX1 expression was observed in LILs and blood cells. In vivo silencing of RUNX1 markedly reduced CD45+CD3+ T cells in LILs as compared to controls (41.5 + 2.5 vs 4.5 + 1.8%). However, CD11c+ dendritic cells were reduced in both LILs and circulation (14.8 + 1.1 vs 0.4 + 0.001, 22.9 + 2.6 vs 1.7 + 0.05%). Discussion: The study highlights the significance of a novel nanodelivery system for in vivo cell-specific gene silencing. LSEC specific decrease in RUNX1 gene expression reduces their angiogenic and adhesion properties, causing decreased infiltration of dendritic and T cells in liver, reducing inflammation in NASH.

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Liver sinusoidal endothelial cells significantly cooperate in the phagocytic and adaptive T cell responses in an experimental model of cirrhosis

Prof Rubén Francés, E Caparrós1, O Juanola1, P Piñero2, I Gómez-Hurtado3, R Linares1, P Boix2, R García-Román1, J Gracia-Sancho3,4, R Francés1,2,3. 1Grupo de Inmunobiología hepática e intestinal, Dpto. Medicina Clínica, Universidad Miguel Hernández, San Juan, Spain. 2Fundación ISABIAL-FISABIO, Hospital General Universitario de Alicante, Alicante, Spain. 3CIBERehd, Instituto de Salud Carlos III, Madrid, Spain; 4Liver Vascular Biology Research Group, IDIBAPS, Barcelona, Spain. Introduction. Cells of the hepatic immune system play a fundamental role both in the detection and direct elimination of antigens, and in the activation of the adaptive response necessary for the maintenance of tissue homeostasis. Aims. To determine the contribution of liver sinusoidal endothelial cells (LSECs) in immune function during experimental cirrhosis. Methods. Untreated Sprague-Dawley rats (Control group) and treated with intragastric CCl4 for 12 weeks (CCl4 group) were included. Perfused livers were collected, and Kupffer (KCs) and dendritic (DCs) cells were purified. LSECs were isolated by differential centrifugation and adhesion on collagen pretreated plates. The phagocytic capacity was measured by internalization of LPS-FITC. For the evaluation of T lymphocytes activation, APCs were preactivated with LPS, and then co-incubated for 48h with autologous CD4+ T lymphocytes isolated from spleen. The phenotypic expression of CD25 and CD71 on CD4+ T lymphocytes was analyzed. Results. The innate immune response in controls is directed by the activity of LSEC cells. Its phagocytic capacity increases in cirrhosis and remains significantly above the phagocytic capacity of DCs and KCs in liver damage conditions (Table 1A). The expression of CD25 and CD71 as T cell activation markers is shown in Table 1B. The ability of induction of specific T cell responses increases in liver damage by all APCs. However, LSECs show an additional activation capacity in response to an acute stimulus in the pre-inflamed cirrhotic environment. Discussion. LSECs participate significantly in the regulation of the immune response in experimental cirrhosis by CCl4.

A)

Control CCl4

DCs 0,289±0,041 0,517±0,187 *

KCs 0,281±0,069 0,604±0,223 *

LSECs 0,416±0,015 0,920±0,820

B)

Control CCl4 Control CCl4

DCs 15,81±10,73 24,74±6,70 1,02±0,14 3,37±1,08 *

DCs+LPS 14,69±7,37 24,44±7,49 * 2,06±1,63 3,90±0,76

KCs 16,55±7,14 24,45±7,96 * 1,47±0,17 4,08±0,92 *

KCs+LPS 16,05±8,27 24,20±7,94 * 2,78±1,33 3,60±0,72

LSECs 17,14±10,68 26,06±3,49 1,83±0,01 3,96±1,48 *

LSECs+LPS 17,26±11,23 35,64±7,84 * 0,92±0,34 9,36±7,00 *

PHAGOCYTOSIS E.coli

abs 450 nm

% cell membrane expression of

CD25 in T cells

% cell membrane expression of

CD71 in T cells

Table 1. A) E.coli phagocytosis by hepatic APCs (absorbance at 450 nm). B)

Cell membrane CD25 and CD71 expression percentage in TCD4+ lymphocytes

coincubated with the different hepatic APCs, preincubated or not with LPS for

18h. * p<0.05 compared to control

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107

Matrix stiffness, a key modulator of liver cells’ phenotype

Dr Sergi Guixé-Muntet1, Martí Ortega-Ribera2, Cong Wang1, Philipp Kellmann1, Jaime Bosch1,2,3, Jean‐François Dufour1,3, Annalisa Berzigotti1,3, Jordi Gracia-Sancho1,2,3. Department of Biomedical Research, University of Bern1, Switzerland; Liver Vascular Biology Research Group, IDIBAPS, CIBEREHD2, Barcelona, Spain; Hepatology, University Clinic for Visceral Surgery and Medicine, Inselspital3, Switzerland. Introduction. During the progression of chronic liver disease (CLD), parenchymal and sinusoidal cells suffer profound modifications in their phenotype ultimately leading to liver microvascular dysfunction, increased intrahepatic vascular resistance and portal hypertension (1). Organ stiffening has been usually understood as a result of stellate cells activation and extracellular matrix deposition during fibrosis development. We do now hypothesize that matrix stiffness might not only be a consequence of disease progression but a key stimulus and a leading cause for liver cells deregulation. Aims. We aim at elucidating the effect of matrix stiffness in modulating the phenotype of the main hepatic cells. Methods. Primary liver cells (hepatocytes: Hep; stellate cells: HSC; and liver sinusoidal endothelial cells: LSEC) were isolated from healthy rats and cultured for 72h on polyacrylamide gels with different stiffness mirroring healthy (0,5kPa), fibrotic (13,5kPa) and cirrhotic (30kPa) liver matrices. Cells’ phenotype was assessed by quantitative morphology, mRNA and protein expression of specific markers and functional assays. Results. Healthy liver cells spontaneously de-differentiated proportionally to stiffness. Specifically, cells at 30kPa displayed larger area, altered morphology, worsened mRNA expression of activation markers [Hep: hepatocyte nuclear factor 4α, albumin; HSC: collagen I, α-smooth muscle actin (α-SMA); LSEC: laminin b1, eNOS] and significantly impaired functionality (Hep: -80% albumin and urea synthesis, HSC: +110% α-SMA fiber formation, LSEC: -40% nitric oxide synthesis and -30% fenestrae porosity and frequency) compared to cells cultured on 0.5kPa gels. Discussion. Matrix stiffness is a key factor in the regulation of liver cells’ phenotype. Stiff substrates, such as those found in advanced CLD environments, favour hepatocyte dedifferentiation, HSC activation and LSEC capillarization. Thus, targeting liver stiffness may represent a novel therapeutic strategy for CLD management. (1) Gracia-Sancho J, Marrone G & Fernández-Iglesias A (2018) Nature Reviews Gastro & Hepatology 16(4):221-234

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Opioid growth factor receptor-like 1; a common mediator of regeneration of fibrotic liver and fetal development of hepatic progenitor cells Assistant Professor Takayo Yanagawa1,2, Hideaki Sumiyoshi1,2, Sachie Nakao1,2, Hiromi Miura1,3, Masato Otsuka1,3, Hiromi Chikada1,3, Akihide Kamiya1,3, Hiroaki Yokomori4, Takashi Takaki5, Yutaka Inagaki1,2. Center for Matrix Biology and Medicine, Graduate School of Medicine1, Department of Innovative Medical Science2, Department of Molecular Life Sciences3, School of Medicine, Tokai University, Isehara, Japan, Department of Internal Medicine, Kitasato University Medical Center4, Kitamoto, Japan, Division of Electron Microscopy, Showa University5, Tokyo, Japan.

Introduction. We have previously identified opioid growth factor receptor like-1 (OGFRL1) as a novel bone marrow cells-derived accelerator of fibrotic liver regeneration. Subsequent studies revealed that OGFRL1 was transiently detected in the circulating blood as an extracellular vesicle (EV) protein upon acute liver injury. Aims. In the present study, we tried to elucidate the mechanisms by which OGFRL1 signal transmitted from the blood cells to the parenchymal hepatocyte accelerates regeneration of the injured/fibrotic liver. Methods. Presence of OGFRL1 protein in liver tissue and isolated cells was detected by protein blot analysis, immunohistochemical staining, and immunoelectron microscopy. Mobilization and proliferation of hepatic progenitor cells (HPCs) in the regenerating fibrotic liver were examined by immunostaining of alpha-fetoprotein (AFP) and BrdU, respectively. HPCs and hematopoietic progenitor cells were isolated from the E13.5 fetal liver, and HPCs were infected with OGFRL1-expressing retroviruses. Results. Administration of OGFRL1-expressing cells into fibrotic mice significantly increased the number of AFP-positive proliferating HPCs after partial resection of the fibrotic liver. In the E13.5 fetal liver, OGFRL1 was predominantly detected in the hematopoietic progenitor cells, and HPCs expressed little, if any, OGFRL1 protein. Overexpression of OGFRL1 in fetal HPCs significantly increased gene expression levels of several hepatocyte-specific markers, such Hnf4a, Cyp2f2, and Fabp1. Discussion. OGFRL1 represents a novel crosstalk between blood cells and parenchymal hepatocytes in both tissue repair and embryonic development. Administration of OGFRL1-expressing cells or OGFRL1-containing EVs could be a potential regenerative therapy for advanced liver fibrosis through mobilization of HPCs.

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Orphan Nuclear Receptor NR4A1: A novel key regulator of Hepatic stellate cell activation

Dr Dinesh Mani Tripathi1,2, Marina Vilaseca1, Erica Lafoz1, Hector Garcia-Caldero1 Savneet Kaur2 Abhishak Gupta2, Sarin SK,2,3 Jaime Bosch1 ,Jordi Gracia-Sancho1 , Juan Carlos Garcia-Pagan1 1Barcelona Hepatic Hemodyanimic Lab, Hospital Clínic, IDIBAPS, CIBEREHD, Barcelona, Spain 2Department of Hepatology & 3Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India Introduction: Hepatic stellate cell (HSC) activation mediates its transition from quiescent to highly proliferative & extracellular matrix producing phenotype, promoting fibrosis. Several fibrogenic pathways and mediators exist and have been deeply investigated. Orphan nuclear receptors represent a family of transcription factors responsible for the regulation of intracellular pathways in many diseases, such as cancer, metabolic and proliferative diseases. Aims: This study aimed to evaluate and understand the role of the orphan nuclear receptor NR4A1 in HSC activation and liver fibrosis. Methods: NR4A1 expression was assessed in healthy and cirrhotic human liver tissues, and in healthy and cirrhotics rats. In addition, HSC were isolated from healthy rats, activated in vitro up to 3 passages (p0, p2, p3) and treated with the NR4A1 agonist, cytosporane B (CsnB; 10 µg/ml) or vehicle (Veh; DMSO) for 24 h. HSC activation markers (αSMA, PDGFRβ and col1α1) and NR4A1 expression were analysed by qPCR, total NR4A1 and phosphorylated/inactivated (pNR4A1) protein expression was analysed by Western Blot. Results: In cirrhotic livers, we observed significantly increased NR4A1 protein expression compared to healthy, in human (+200-350%) and in rats (TAA: +20%; CCl4: +49%). However, increment was mainly due to phosphorylation of NR4A1 (pNR4A1, TAA: +11%; CCl4: +57%), suggesting a pathological inactivation of this transcription factor.In vitro, significant induction of NR4A1 during HSC activation. Indeed, NR4A1 mRNA expression was significantly increased from p0 to p2 (>8 fold), and in p3 (>30 fold), in comparison to p0 HSC. Interestingly, increased NR4A1 positively correlated with HSC activation markers (αSMA, Col1a1 and PDGFBR). Pharmacological activation of NR4A1 by CsnB was capable to prevent HSC de-differentiation as demonstrated by significant down-regulation of the HSC activation markers αSMA (-93% and -97%), PDGFRβ (-84% and -86%) and Col1a1 (-49% and -75%) in comparison to vehicle-treated cells in p2 and p3, respectively. Discussion: Our study demonstrates for the first time NR4A1 as a novel modulator of HSC phenotype. Interestingly, inactive form of this transcription factor is highly expressed in cirrhotic livers.Thus we herein propose modulation of NR4A1 activity as a promising avenue to treat chronic liver disease.

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Old age and the liver sinusoid

Prof David Le Couteur, Victoria Cogger The University of Sydney, Australia Old age is associated with changes in the cells of the hepatic sinusoid, including defenestration of the liver sinusoidal endothelial cells, low grade activation of Kupffer cells, and accumulation of lipid droplets in hepatic stellate cells. Fenestrations in liver endothelial cells provide a portal for the transfer of substrates such as lipoproteins and insulin between the hepatocytes and sinusoidal blood. Age-related defenestration may therefore contribute to age-related changes in circulating lipoproteins and hepatic insulin sensitivity. Drugs that target fenestrations in old age may be useful in the treatment and prevention of dyslipidemia and insulin resistance. Nanoparticles such as quantum dots show promise for targeting liver endothelial cells, specifically delivering fenestration-active agents to the liver sinusoidal endothelial cells.

111

Sinusoidal cells in ALD

Dr Adam Kim1

1Liver Center, Cleveland Clinic, United States

Alcohol-related Liver Disease (ALD) is a spectrum of pathologies where the innate immune system contributes significantly to liver damage. Kupffer cells, the resident macrophage of livers, are critical to the progression of (ALD). In response to chronic ethanol, they become sensitized to bacterial lipopolysaccharides (LPS) and express more pro-inflammatory cytokines and markers for macrophage polarization. Here we use a next-generation sequencing to understand why innate immune cells become hypersensitive to LPS in ALD. First, we use miRNA sequencing to understand the spectrum of macrophage polarization after chronic ethanol feeding in rats. Differential expression analyses revealed 40 misregulated miRNAs in Kupffer cells from the chronic ethanol-fed rats compared to pair-fed (FDR<0.2). 23/40 miRNAs clustered in 13 distinct regions of the genome. Each cluster contained at least 3 differentially expressed miRNAs, where 12/13 clusters had decreased expression in chronic ethanol. Five downregulated clusters contained miRNAs with known roles in polarization, including miR125a, let-7c, miR221, and miR222. Correlation analyses revealed that miRNAs in three of these clusters were co-regulated and located within the introns of host genes, implying co-transcriptional regulation. Similar to the rat Kupffer cells, peripheral blood mononuclear cells (PBMCs) isolated from alcoholic hepatitis patients were also hypersensitive to LPS. Additionally, we observed dysregulation of a polarization genes and polarization-associated miRNAs, indicating a highly heterogeneous M1/M2 phenotype. Together, these data implicate a role for dysregulated miRNAs in the spectrum of macrophage polarization.

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112

Sinusoidal cells in HCC and NASH

Prof Jean-François Dufour University Clinic for Visceral Surgery and Medicine, Inselspital, Switzerland

Non-alcoholic steatohepatitis is an inflammatory metabolic disease of the liver. Therefore, the cell types which attracted most research attention are the hepatocytes, the Kupffer cells and the stellate cells. However, patients with NASH die primarily from cardiovascular complications suggesting than vessels and endothelial cells are involve in the pathophysiology of the disease. Indeed, hepatic sinusoidal cells are dysfunctional in NASH: they lose their fenestrae, they secrete inflammatory mediators and produce less vasodilators. This changes promote steatosis and the recruitment in the parenchyma of inflammatory cells. Restoring a normal endothelial cell phenotype is a therapeutic option to improve NASH and reduce its carcinogenic risk.

113

Post-translational modifications drive the effects of high-mobility group box-1 during liver fibrosis progression and resolution

Prof Natalia Nieto1

1University of Chicago, Chicago, United States

Background & Rationale: damage-associated molecular patterns (DAMP) play a major role in chronic liver disease. We previously demonstrated that High-Mobility Group Box-1 (HMGB1) is significantly increased and undergoes post-translational modifications (PTMs) in response to hepatic injury; thus, we hypothesized that specific HMGB1 isoforms could participate in the progression and/or resolution of liver fibrosis by regulating scarring and the hepatic stellate cell (HSC) fate. Results: using electrospray ionization-liquid chromatography-mass spectrometry (ESI-LC-MS) we identified the signature oxidant stress-sensitive HMGB1 isoforms in serum and liver from patients and in mice with liver fibrosis. Hepatocytes and to a lesser extent myeloid cells were the main source of these isoforms in carbon tetrachloride (CCl4)-induced liver fibrosis progression and resolution. Besides fully reduced ([H]), disulfide ([O]) HMGB1 rose during fibrosis progression and declined during fibrosis reversal; yet, sulfonate ([SO3]) HMGB1 appeared only in the resolution phase and was significantly increased. [O] and [SO3] HMGB1 were produced by hepatocytes. Injection of [O] HMGB1 increased liver fibrosis in mice more than injection of [H] HMGB1. Likewise, ablation of [O] HMGB1 in hepatocytes reduced CCl4-induced liver fibrosis. The pro-fibrogenic effect of [H] and [O] HMGB1 in HSC was mediated by the Receptor for Advanced Glycation End-Products (RAGE) in vitro and in vivo. In the resolution phase, the anti-fibrogenic effect of [SO3] HMGB1 was due to induction of HSC apoptosis mediated by RAGE activation. Conclusions: the redox-sensitive dynamic changes in the HMGB1 isoforms signal via RAGE-dependent mechanisms to drive the HSC pro-fibrogenic phenotype and fate, therefore contributing to the progression and/or resolution of liver fibrosis. Consequently, they could be used as biomarkers for prognosis, the clinical course of liver fibrosis and the therapeutic response.

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114

Treatment of chronic liver diseases using a rationally designed protein that induces apoptosis by targeting integrin avb3

Zhi-Ren Liu and Chakra Ravi Turaga Department of Biology, Georgia State University, Atlanta, GA, USA Introduction. Chronic Liver Diseases (CLD) are the results of inflammatory responses to liver insults, which lead to abnormal accumulation of collagen fibrils, neo-angiogenesis, and sinusoidal remodeling in the fibrotic liver. Collagen deposition along with intrahepatic angiogenesis and sinusoidal remodeling alters sinusoid structure result in portal hypertension, liver failure, and other complications. Efforts were made to develop treatments for CLD and fibrosis. However, the success of such treatments is limited and unpredictable. There is unmet medical need to develop effective treatment for CLD and associated complications. Aims. To develop effective treatment drug for CLD by targeting activated hepatic stellate cells (HSC) and capillarized liver sinusoidal endothelial cells (LSEC). Methods. We report a strategy and a drug candidate for CLD treatment by induction of integrin v3 mediated cell apoptosis using a rationally designed protein (ProAgio) with mouse models of liver fibrosis and cirrhosis. ProAgio is designed to target integrin v3 at a novel site and induces apoptosis of the integrin v 3 expressing cells. Integrin v3 is highly expressed in activated HSC, angiogenic endothelium, and capillarized LSEC. Results. ProAgio induces apoptosis of activated HSC and capillarized LSEC in vitro. Tests with liver fibrosis/cirrhosis mouse models demonstrate that ProAgio reverses liver fibrosis/cirrhosis and relieves blood flow resistance by depleting activated HSC and capillarized LSEC in fibrotic liver. ProAgio treatment resorbs collagen (including reduces collagen crosslinking), abrogates intrahepatic angiogenesis, and reverses sinusoidal remodeling in fibrotic liver. Discussion. Our studies demonstrate that induction of integrin v3 mediated apoptosis by the engineered protein drug is a novel and effective approach for CLD treatment. Simultaneously depletion of both activated HSC and capillarized LSEC by ProAgio in fibrotic liver certainly brings a very important advantage of breaking down the vicious cycle of angiogenesis-fibrogenesis in treatment of CLD. ProAgio may also be an effective treatment agent for hepatic portal hypertension associated with CLD progression.

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115

5-Hydroxytryptamine receptor 1A, a novel therapeutic target for portal hypertension

Dr Chang-Peng Zhu1, Wei-Fen Xie1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University1, Shanghai, China. Introduction. Portal hypertension (PH) is a major complication of chronic liver disease. It is known that serum 5-hydroxytryptamine (5-HT) is increased in patients with PH, but the role of 5-HT and its receptors in PH remains unknown. Aims. This study aims to evaluate the pathophysiological role of 5-HT receptor 1A (HTR1A) in the hepatic and systemic hemodynamic, and liver fibrosis, in rats with chronic liver disease. Methods. Thioacetamide (TAA) and carbon tetrachloride (CCl4) were used to induce liver cirrhosis in rats. HTR1A selective agonist (8-OH-DPAT) and antagonist (WAY-100635) were used to determine the effect of HTR1A in PH. Results. The expression of HTR1A in portal veins (PVs) was elevated in cirrhotic rats and patients. The portal pressure (PP) was significantly elevated by intra PV infusion of either 5-HT or 8-OH-DPAT in normal rats, which was inhibited by HTR1A antagonist WAY-100635. Moreover, one-week administration of 8-OH-DPAT remarkedly increased PP in both TAA model and CCl4 model rats (TAA: 8.59 ± 1.31 cmH2O vs 12.18 ± 1.90 cmH20, P < 0.001; CCl4: 8.47 ± 1.27 cmH2O vs 11.66 ± 1.89 cmH20, P < 0.01). In contrast, WAY-100635 decreased PP by 37% in both TAA and CCl4 models (TAA: 21.07 ± 2.27 cmH2O vs 13.27 ± 3.00 cmH20, P < 0.001 (accompanying figure); CCl4: 11.85 ± 3.22 cmH2O vs 7.36 ± 2.14 cmH20, P < 0.05), which was comparable to carvedilol (TAA: 14.01 ± 4.14 cmH2O; CCl4: 7.99±1.37 cmH2O). Meanwhile, hepatic fibrosis was not alleviated by one-week delivery of WAY-100635. Interestingly, no significant changes of mean arterial pressure and heart rates were found in WAY-100635 group, while carvedilol treatment resulted in hypotension and bradycardia in cirrhotic rats. Discussion. 5-HT plays a critical role in the pathogenesis of PH. HTR1A served as a novel therapeutic target for PH. The selective HTR1A antagonist WAY-100635 may have advantage in the treatment of PH when compared with carvedilol as it has no obvious systemic side effects.

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116

Effect of Candesatran cilexetil on the regulation of hepatic NOSTRIN-eNOS pathway in cirrhosis

Dr Vairappan Balasubram, Sundhar M1, Kamalakkannan V2, Ravikumar T.S1 and Wright G3. 1Liver Disease Research Lab, Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry-605006, India; 2Technology absorption group, Strides Shasun Limited, Pondicherry-605014, India; 3Royal Free Hospital, London, UK.

Introduction. In decompensated cirrhosis, severity of portal hypertension (PHT) is associated with increased hepatic endothelial nitric oxide synthase (eNOS) trafficking inducer (NOSTRIN); although the mechanism remains unclear. Aims. Establish whether in cirrhosis-PHT models, superimposed inflammation (to mimic acute-on-chronic liver failure - ACLF) modulates hepatic NOSTRIN expression, nitric oxide (NO) synthesis and/or endothelial dysfunction (ED). Secondarily, whether the ‘Angiotensin II type-1 receptor blocker’ Candesartan Cilexetil (CC), effects this pathway. Methods. 4-weeks after Bile-duct ligation (BDL) or sham-operation, BDL rats were randomized 3-hours prior to sacrifice, to receive either lipopolysaccharide (LPS; 1 mg/kg, i.p.) or saline. In addition, CD-1 mice received carbon tetrachloride injections (CCl4 15% v/v corn oil, 0.5 ml/kg, twice-weekly i.p.) for 13-weeks to induce cirrhosis. After 3-months, mice were randomized to 2-weeks oral administration of CC (8mg/kg) or DMSO ± LPS. In both models at sacrifice, plasma (biochemical, cytokines and angiotensin II) and liver tissues (histopathology and Sirius-red stains) were analysed. Results. When compared to controls, BDL and CCl4 animals showed markedly elevated hepatic gene and protein NOSTRIN expression (p<0.0001), whilst hepatic eNOS activity was significantly reduced (p<0.05). LPS-challenge to cirrhotic animals further increased NOSTRIN and reduced eNOS levels (p<0.05, respectively). In BDL animals portal pressure was significantly increased compared to sham and was unchanged with LPS-challenge (14 ± 1.2 vs. 5.8 ± 0.9 mmHg; p<0.0001). On confocal microscopy NOSTRIN and eNOS was primarily within hepatic vascular endothelial cells. CC-treatment to CCl4±LPS showed significantly reduced NOSTRIN (p<0.05) and increased hepatic NO (p<0.01). eNOS, iNOS and caveolin-1 protein expressions were significantly increased in CCl4 animals. CC-treatment non-significantly lowered eNOS/iNOS expression; although caveolin-1 expression was unaltered. Discussion. This study is the first to indicate a potential mechanistic association between NOSTRIN-NO pathways in cirrhosis and ACLF development. Moreover, this pathway provides a potential therapeutic target given the ameliorative response to Candesartan treatment.

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117

LSEC phenotype restoration by statins reduces portal pressure in a rat NASH model

Miren Bravo M1,2, Imma Raurell1, Salvador Augustin1,2, Joan Genescà1,2 and María Martell1,2.

Institut de Recerca Vall d’Hebron (VHIR)1, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD)2, Madrid, Spain. Introduction. Liver sinusoidal endothelial cell (LSEC) dysfunction has been associated with early stages of liver diseases including non-alcoholic steatohepatitis (NASH), a worldwide spreading chronic disease. Yet, their dedifferentiation into a pathologic phenotype has not been entirely assessed in this etiology. Statins have been shown to ameliorate endothelial function, modulating non-parenchymal cells phenotype and sinusoidal resistance and thus improving portal hypertension (PH). Aims. We aimed to characterize LSEC dedifferentiation and its contribution to PH in NASH, together with elucidating statins usefulness in restoring the healthy phenotype of these cells in a rat model of NASH with PH. Methods. Sprague-Dawley rats fed with high fat glucose-fructose diet (HFGFD), or control diet (CD) for 8 weeks were treated with simvastatin (sim) (10mg·kg-1·day-1), atorvastatin (ato) (10mg·kg-1·day-1) or vehicle during 2 weeks. LSEC from these animal groups were sorted as differentiated (CD32b+) or dedifferentiated (CD32b−) and characterized individually by RT-qPCR. Hepatic stellate cells (HSC) from the same groups were analysed by immunofluorescence and WB. Biochemical, histological and hemodynamic determinations were also carried out. Results. HFGFD rats developed obesity, insulin resistance and histological NASH (defined as NAFLD activity score ≥3) without fibrosis. They showed increased portal pressure (PP) compared with CD rats (10.47±0.37mmHg vs 8.30±0.22mmHg; p<0.001), most likely due to endothelial dysfunction as confirmed by decreased hepatic P-AKT and P-eNOS, a higher percentage of dedifferentiated (CD32b−) LSEC (8% vs 1%; p=0.005) and increased levels of endothelin-1 (ET-1). HSC from HFGFD animals lost vitamin A storage and displayed activation of ET-1 downstream signaling. Statins caused a reduction in PP (sim: 9.29±0.25mmHg, p<0.01; ato: 8.85±0.30mmHg, p<0.001), as well as NASH reversion and a significant recovery of LSEC differentiation leading to a more quiescent phenotype of HSCs. Discussion. LSEC dedifferentiation is responsible for the contractile sinusoidal profile leading to portal hypertension in early NASH, while statins confer vasoprotection through LSEC functional phenotype recovery and HSC deactivation, reinforcing the therapeutic potential of these drugs for NASH.

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118

Angiotensin and cyclooxygenase-mediated mechanisms attenuate the increased transhepatic pressure gradient in rats with severe steatosis

Denise van der Graaff1,2, Wilhelmus J Kwanten1,2, Peter P Michielsen1,2, Joris G De Man1, Benedicte Y De Winter1, Sven M Francque1,2. Laboratory of Experimental Medicine and Pediatrics, University of Antwerp1, Antwerp, Belgium; Department of Gastroenterology and Hepatology, Antwerp University Hospital2, Edegem, Belgium. Introduction. The intrahepatic vascular resistance (IHVR) is increased in early non-alcoholic fatty liver disease (NAFLD), impairing hepatic blood flow and potentially causing tissue hypoxia and disease progression. Aims. The aim was to elucidate the effects of angiotensin II (ATII) and cyclooxygenase (COX) on the IHVR in a model of severe steatosis. Methods. The IHVR was studied by measuring the transhepatic pressure gradient (THPG) in an in situ ex vivo perfusion model. The THPG was studied in male Wistar rats (n≥7/group) fed a methionine-choline-deficient diet, which induces severe steatosis after 4 weeks, or a control diet. The effects of Valsartan (VAL, an ATII receptor blocker, 0.2-60 µM) were tested in dose-response experiments, increasing the dose by 0.5 log M every 5 minutes at a constant flow after pre-constriction by 0.01 µM ATII. Likewise, SC-560 (a COX-1 inhibitor, 0.03-10 µM) and SC-236 (a COX-2 inhibitor, 0.003-1 µM) were tested in dose-response experiments after pre-constriction by α1-adrenergic agonist methoxamine (Mx, 1 µM). Results. Blocking the ATII receptor attenuated the ATII-induced increase in THPG, thereby normalising the THPG in steatosis comparable to controls from the lowest dose onwards (controls: 10.1±0.7 to 7.8±0.6 mmHg; steatosis: 12.5±0.5 to 9.6±0.4 mmHg; ATII vs ATII+6 µM VAL, p<0.001). COX-1 inhibition did not significantly affect the THPG, whereas COX-2 inhibition significantly attenuated the increased THPG in steatosis but not in controls (controls: 9.3±0.9 to 8.6±0.4 mmHg (p=0.38); steatosis: 14.9±1.1 to 11.2±0.5 mmHg (p<0.01); Mx vs MX+ 1 µM SC-236)(Figure). Discussion. Blocking ATII and COX-2 receptors mitigated the increased THPG in severe steatosis. Therefore, ATII- and COX-mediated pathways seem to be involved in the increased IHVR in NAFLD and could potentially be interesting therapeutic targets in the treatment of NAFLD.

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119

NOX2 activation by the aging protein p52Shc leads to accelerated fibrosis in older mice

Prof Natalie Torok, Sarah Fish1, Alexey Tomilov2, Ali Dehnad3, Joy Jiang2, Suvarthi Das3, Jon Ramsey2, Gino Cortopassi2, Natalie J. Torok3 1UC Davis Medical Center, Sacramento, CA; 2School of Veterinary Medicine, and Molecular Biosciences, UC Davis, Davis, CA, and 3Stanford University, CA Introduction. Clinically, older patients present with advanced fibrosis in non-alcoholic steatohepatitis (NASH). Whether this is due to activation of aging-related molecular pathways, is unknown. The Src homology 2 domain containing (Shc) family of proteins have been recognized in aging however, their link redox stress and fibrosis in NASH have not been studied. NADPH oxidases (NOXs) are key fibrogenic enzymes, and the phagocytic NOX2 is expressed in hepatocytes but its mode of activation in these cells has not been revealed. Aims. To study if aging induces more NOX2 activation/redox stress via Shc leading to rapid fibrosis/NASH progression. Methods. Shc was studied in liver samples of young and old patients. Shc, NOX2 expression and activation were assessed in young and old mice on fast food diet (FFD) by RTqPCR, IHC and western blots. Shc was inhibited by a lentiviral (LV) shRNA approach in old mice on FFD, and NOX1, 2, 4, ROS, lipid peroxidation, inflammation and fibrosis were studied. To address the role of Shc in a cell-specific manner, fl/flShc mice were injected with AAV8-TBG-cre. The interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by IP, and biolayer interferometry (BLI), using full-length and truncated p52Shc mutants. Results. Shc was induced in older patients, and mice on FFD. Fibrosis (procollagen α1 (I), TGFβ, αSMA, p< 0.05), Shc expression and p52Shc activation (p-Y317) were more induced in aged mice on FFD. NOX2 was induced in a Shc-dependent manner during FFD (p=0.03). Inflammatory (MCP-1, TNFα, IL-1β) and fibrogenic transcripts were blunted; and picrosirius red showed less fibrosis in LV-shShc-injected old mice, and in hepatocyte Shc-deleted mice. They also had attenuated oxidative stress, lipid peroxidation and triglyceride content. IP revealed a novel association between p47phox and p52Shc induced by palmitate, and BLI assays demonstrated the direct binding of p52Shc to p47phox, while deletion mutants for the SH2 and PTB domain produced a much lower response. Discussion. We have revealed a novel mechanism of activation for p47phox/NOX2 in a non-phagocytic manner, by p52Shc in aging hepatocytes. Enhanced p52Shc/NOX2 expression, activation and redox stress during aging contribute to an exacerbated inflammatory and fibrogenic process in NASH that could be therapeutically targeted.

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120

Mediators of inflammation and fibrogenesis associated with hepatic stellate cell biology

Prof Grant A. Ramm, BSc, BSc (Hons), PhD, FAASLD Head of Department – Cell and Molecular Biology, and Principal Research Fellow, QIMR Berghofer Medical Research Institute, Brisbane, Australia. Senior Research Fellow, National Health and Medical Research Council of Australia. In chronic liver disease, hepatic fibrosis occurs when resident myofibroblast precursors (hepatic stellate cells [HSCs] and portal fibroblasts), are activated to produce excessive fibrillar collagen and extracellular matrix. HSCs are non-parenchymal cells located in the sub-endothelial space of Dissé, in close contact with hepatocytes, macrophages, endothelial cells, and in chronic disease, liver progenitor (stem) cells (LPCs). It has been proposed that HSCs are regulated by their paracrine environment via local soluble mediators and direct cell contact with various other liver cell types. Chronic liver diseases are generally characterised by similar cellular and histological processes, involving (i) inflammation, (ii) LPC expansion and (iii) fibrogenesis.

Inflammation is a critical earlier driver of fibrogenesis mediated via NFB Interleukin 1-beta (IL-1) is a pivotal inflammasome-regulated inflammatory mediator of fibrogenesis. Recent work by our group has demonstrated a role for heavy-chain Ferritin (FTH1), released as a result of hepatocellular injury, as a novel damage-associated molecular pattern (DAMP) which activates the NLRP3 inflammasome via ICAM-1, driving HSC-derived IL-1 secretion. Other mediators of fibrogenesis have been identified via biomarker development studies designed for early detection of liver disease or staging of hepatic fibrosis, providing mechanistic clues to the regulation of fibrosis. microRNAs (miRs) identified in such studies have been shown to aid in controlling the collagen synthetic pathway. Our research has identified miR25 as anti-fibrotic ‘brake’ on HSC collagen synthesis by inhibiting regulators of Notch signalling that control TGF R1 expression and thus suppress TGF -induced collagen synthesis. Identification of such driver mechanistic molecules and pathways of fibrosis development will aid in the development of novel therapeutic interventions. Such potential novel therapies designed to control inflammation or collagen synthesis by targeting inflammasome intermediates or regulatory microRNAs could have substantial clinical benefit in mitigating chronic liver disease progression and subsequent sequelae including cirrhosis, portal hypertension, liver failure and hepatocellular carcinoma.

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121 Animal models to study liver diseases Francisco Javier Cubero1,2 1Department of Immunology, Ophthalmology & ORL, Complutense University School of Medicine, Madrid, Spain212 de Octubre Health Research Institute (imas12), Madrid, Spain Liver diseases are frequent and potentially life-threatening for humans. The underlying etiologies range from toxins, autoimmune disorders, alcohol abuse to imbalanced diets. In early stages of disease, the liver regenerates once the insult is gone. However, advanced stages require organ transplantation. Thus, a better understanding of underlying mechanisms is mandatory for the design of new therapeutic avenues that can be translated into the clinics. The generation and exploitation of animal models is not only indispensable to unravel the pathogenesis mechanisms triggering the various liver diseases, but also for the identification of potential targets with theranostic value. Nowadays, rodents are the most frequently used animal model for the study of liver disease. However, animal models have both advantages and drawbacks. Whilst most animal models of liver disease are very useful for elucidating pathophysiology, some do partially represent the characteristics of human liver diseases.. Therefore, classical models of liver fibrosis, cholangitis, alcoholic liver disease (including acute and chronic binge drinking), dietary models that recapitulate human non-alcoholic fatty liver disease (NAFLD) and spontaneous models of chronic liver disease will be thoroughly discussed in order to provide the basis for the selection of the most suitable animal model given the purpose of the research study.

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122

Targeted delivery to the liver sinusoidal endothelium using silver sulfide quantum dots

Nicholas Hunt 1, 2, 3, Glen Lockwood 1, 3, Frank Le Couteur 1, Peter McCourt 3, 4, Nidhi Singla 5, Sun Woo Sophie Kang 1, 3, Zdenka Kuncic 3, 5, 6, David Le Couteur 1, 2, 3, Victoria Cogger 1, 2, 3. 1 ANZAC Research Institute, AAAI and CERA, Concord Repatriation General Hospital; 2 Concord Clinical School,

3 Charles Perkins Centre, 5 School of Physics, 6 Nano Institute, The University of Sydney, Sydney, NSW, Australia; 4 Department of Medical Biology, University of Tromsø, Norway

Silver sulfide quantum dots (QDs) are used in a biological setting for imaging and to transport materials or therapeutics, potentially to very specific cell types within the body. Here we demonstrate targeted delivery of QDs to the liver sinusoidal endothelial cell (LSEC) in vitro and in vivo. Water soluble mono-dispersed silver sulfide QDs with different biopolymers were synthesized. QDs were radiolabelled with 3H-oleic acid in addition to a fluorescent tag placed within a drug binding site. Biodistribution was measured from a single oral gavage of 1 mM QDs in mice using scintillation counting of samples taken from isolated tissues and cells. Flow cytometry was also performed to confirm the delivery of a florescent tag to LSECs relative to other liver cells. Transmission electron microscopy (TEM) imaging and was used to visualise QDs localised to liver sinusoidal endothelial cells with energy loss spectroscopy (EELS) and energy dispersive spectroscopy (EDS) performed. Endocytosis pathways were also investigated using the immortal LSEC cell line SK-Hep1. The prepared QDs were spherical with a mean diameter of 8 nm and a zeta potential of -30. Three different polymer coatings were prepared: formaldehyde treated bovine serum albumin; gelatine; and non-fractionated heparin. Fourier transformed infrared microscopy (FTIR) confirmed the loading of these biopolymers onto QDs, with complete coverage identified by topological changes measured with FTIR. Endocytosis was investigated with the LSEC cell line SK-Hep1. Cells were incubated with 1mM 3H-QDs for 24 hrs with inhibition of endocytosis promoted by 5 mM sucrose pre-treatment demonstrating clathrin mediated endocytosis. Biodistribution studies with gavaged QDs demonstrated that targeting of the liver could be improved by polymer coating from 50% to 70%. Within the intact liver, LSEC preference improved from 35% to 75% uptake. Measurements were confirmed by fluorescent tag localisation to the LSEC population using flow cytometry. Localisation of QDs to LSECs was additionally observed using immunofluorescence, H&E and transmission electron microscopy. In conclusion, we have shown high specificity targeting of the LSEC by QD delivery of a fluorescent tag in a gavaged mouse with the use of biopolymer coatings.

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123

Scavenger function of liver sinusoidal endothelial cells in a transgenic mouse model of liver fibrosis is reduced

Karen K. Sørensen1, Milton B. Antwi1, Gianina Dumitriu1, Ruomei Li1, Bård Smedsrød1, Winnie Eskild2. 1Department of Medical Biology, UiT The Arctic University of Norway1, Tromsø, Norway; 2Department of Biosciences, University of Oslo, Oslo, Norway Introduction: The liver sinusoidal endothelial cells (LSEC) are important scavenger cells that daily remove large amounts of waste macromolecules and antigens by receptor-mediated endocytosis. However, our knowledge about how this function is affected in liver disease is still limited. The transgenic Glmpgt/gt mouse [1, 2] lacks the expression of a lysosomal membrane protein, GLMP (Glycosylated Lysosomal Membrane Protein), and develops liver fibrosis as its dominating phenotype. The mice are born normal, grow well and have normal fertility, but develop liver fibrosis at 3-4 months of age. The liver pathology progresses into cancer after 12-18 months. Aims. The aim of the study was to characterize LSEC endocytic function, receptor expression and ultrastructure in the Glmpgt/gt mouse model of slowly progressing fibrosis. Methods. A ligand for the LSEC scavenger receptors, FITC-FSA, was administered i.v. into Glmpgt/gt mice and Glmpwt/wt control mice (4 months and 10 months; both genders). Tissues were harvested after 10 min and prepared for light microscopy, IHC, and electron microscopy. Quantitative image analyses were done using Fiji (image J). Results. Uptake of FITC-FSA in sinusoids was markedly and significantly reduced in the Glmpgt/gt livers and showed an inverse relationship with collagen content in sections. The expression of LSEC endocytosis receptors was variable. Scanning electron microscopy showed intact LSEC fenestrations and typical sieve plates in most hepatic sinusoids in the Glmpgt/gt mice. Destruction of LSECs were only observed in the most injured areas. Discussion. Liver fibrosis was found to be correlated with reduced LSEC scavenger function. As opposed to what is reported in other models of liver fibrosis, LSEC fenestrations were well preserved, also in some areas with high collagen content. This suggests that LSEC defenestration in liver fibrosis is caused by the underlying disease. Furthermore, our results indicate that LSEC scavenger function and fenestration are independent variables. 1. Kong XY et al (2014) Dis Model Mech, 7(3): p. 351-62. 2. Nesset CK et al (2016) Fibrogenesis Tissue Repair, 9: p. 5.

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124

Liver stiffness as a target factor for advanced chronic liver disease: Unstretching the nucleus

Martí Ortega-Ribera1, Sergi Guixé-Muntet2, Cong Wang2, Philipp Kellmann2, Jaime Bosch1,2,3, Jean‐François Dufour2,3, Annalisa Berzigotti2,3, Jordi Gracia-Sancho1,2,3. Liver Vascular Biology Research Group, IDIBAPS, CIBEREHD1, Barcelona, Spain; Department of Biomedical Research, University of Bern2, Switzerland; Hepatology, University Clinic for Visceral Surgery and Medicine, Inselspital3, Switzerland. Introduction. Liver stiffness is an accurate diagnostic parameter in advanced chronic liver disease (ACLD) and it is being currently studied in vitro not only as a consequence of fibrosis progression but as a central factor in the deregulation of healthy liver cells. Therefore, we hypothesize that the stiffness-mediated activation of hepatic cells could play a driving role in ACLD and represent a novel target for its treatment. Aims. We aimed at 1-characterizing the role of matrix stiffness on the phenotype of cirrhotic cells, 2-assessing its influence on their response to anti-fibrotic strategies and 3-determining the stiffness-mediated molecular mechanism. Methods. Primary liver cells (hepatocytes; stellate cells: HSC, and endothelial cells: LSEC) were isolated from CCl4-cirrhotic rats and cultured for 72h on polyacrylamide gels with decreasing physiological stiffnesses. Cells’ phenotype was assessed by morphology, mRNA and protein analyses. The response of cirrhotic HSC to anti-fibrotic drugs (50µM liraglutide and 10µM simvastatin) was determined in stiff vs soft matrices. Nuclear deformation in response to stiffness was quantified in cells treated with cytoskeleton disruptors (2µM Cytochalasin D + 1µM Nocodazole) or veh. Results. Cirrhotic cells displayed a significant amelioration in all the assessed phenotype markers when cultured on soft surfaces compared to stiffer ones (e.g. hepatocytes: +500% albumin synthesis; HSC: -70% α-SMA protein, LSEC: +70% NO and fenestrae). Liraglutide showed -50% effectiveness on HSC at low vs high stiffness, while simvastatin had similar antifibrotic effects on both matrices. Finally, high stiffness induced a >3-fold deformation in the nuclei of hepatocytes, HSC and LSEC. However, inhibition of cytoskeletal tension significantly recovered spherical nuclear shape and expression of phenotype markers in cells on stiff surfaces. Discussion. Stiffness modulates the phenotype of cirrhotic cells and their response to antifibrotic drugs by altering the nuclear morphology through the cytoskeleton. This mechanism may be fully reversible, which suggests that targeting the stiffness-derived intracellular mechanical tensions may be a novel and promising therapeutic strategy for the treatment of ACLD. Additionally, stiffness may determine the effectiveness of current therapeutic options for ACLD.

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125

Matrix stiffness regulate Liver Sinusoidal Endothelial Cells (LSECs) function: Importance for Liver Fibrosis Progression

Assoc Prof Srivatsan Kidambi1, Vaishaali Natarajan1, Carol Casey2, Edward Harris3 1Department of Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA; 2Department of Internal Medicine Division of Gastroenterology-Hepatology, University of Nebraska Medical Center, Omaha, NE, USA; 3Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, USA

Introduction. Liver sinusoidal endothelial cells (LSECs) are a highly specialized endothelial cell that participates in numerous liver metabolic activities and collectively function as a scavenger system. Prior to hepatic fibrosis, independent of their etiology, LSECs become highly pro-inflammatory, capillarized (loss in fenestrations), and loss in specialized receptors (Stabilin-1, Stabilin-2, CD31 and SE-1). Liver fibrosis also leads to significant loss in the endocytosis function of LSECs. Thus understanding regulation of LSEC phenotype may be critical to understanding fibrosis. Extensive remodeling of the extracellular matrix occurs during fibrosis that leads to liver stiffening. The role of matrix stiffness as related to subtle but pivotal changes in LSECs physiology is under explored. Aim. Investigate the role of stiffness in modulating LSECs function in fibrotic-like microenvironment using our innovative biomimetic liver fibrosis model that allows modulation of substrate stiffness. Methods. Primary LSECs were cultured on our novel polymer film coated polydimethylsiloxane (PDMS) gels with 2 kPa, 9 kPa 25 kPa and 55 kPa elastic modulus mimicking healthy, early fibrotic, fibrotic and extremely fibrotic substrates. SEM was used to image to fenestrations of LSECs and HA endocytosis assay to measure the LSECs function. Results. LSECs cultured on stiffer environment had significant remodelling of cytoskeletal proteins and morphology indicated of stress fibers. Also we observed that LSECs on fibrotic substrates resulted in loss of fenestrations. This is critical as loss in fenestrations has been show to precede hepatic fibrosis and these LSECs lose the ability to promote hepatic stellate cell (HSC) quiescence. LSECs on stiffer environment also had higher expression of cell adhesion molecules, VCAM-1 and ICAM-1 indicating the loss of phenotype of the cells. Fibrotic stiffness also impeded the HA endocytosis in LSECs, one of the main functions of the cells. Similar effect was observed in LSECs isolated from Non-Alcoholic Fatty Liver Disease (NAFLD) rat models indicating correlation to physiological conditions. Conclusions. Together, all these data demonstrate the plausible role of stiffness in regulating LSECs function and contribute to HSC activation and progression of liver fibrosis.

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126

Polymeric micelles as a drug delivery system targeting liver sinusoidal endothelial cells in chronic liver disease: Validation with simvastatin encapsulation

Diana Hide1,2, Mar Gil1, Fernanda R. Andrade3, Diana Rafael3, Salvador Augustin1,2, Simo Schwartz Jr3, Joan Genescà1,2, and Maria Martell1,2. Liver Unit, Institut de Recerca Vall d’Hebrón (VHIR)1, Barcelona, Spain; Centro de Investigación Biomédica en Red (CIBER)2, Madrid, Spain; Drug Delivery and Targeting group, CIBBIM3, Barcelona, Spain. Introduction. Liver sinusoidal endothelial cells (LSEC) are unique among endothelial cells and play an essential role during the initiation of any chronic liver disease (CLD). However, there are no specific treatments to target this cell type. Although statins have demonstrated high potential to treat chronic liver disease (CLD) its side effects in patients with severely deteriorated hepatic function limit the administered dose and, thus, its effect. Aims. To develop a drug delivery system based on polymeric micelles (PM) to better target LSEC and encapsulate simvastatin (PM-simva) as proof of concept in order to improve its effectivity and safety. Methods. Physicochemical characterization of PM (drug encapsulation, size, and polydispersion index); functional characterization in LSEC, hepatic stellate cells (HSC), Kupffer cells (KC) and hepatocytes isolated from control and bile duct ligated (BDL) rats comprised internalization, cytotoxicity, gene expression assays and phenotype examination by scanning electronic microscopy. In vivo administration was performed for biodistribution assays. Results. The produced PM were very homogeneous with 22.4nm average diameter and simvastatin entrapment efficiency >96%. In vitro they were highly internalized by healthy LSEC (80% positive cells) and HSC (70%) followed by far by hepatocytes and KC. LSEC derived from cirrhotic rats maintained their internalization capacity but in HSC was dramatically reduced from 70 to 30%. PM-simva were less toxic that the non-encapsulated drug in all liver cell types. Functionally, the effects of PM+simva were similar to free simvastatin: KLF2 was overexpressed in all cell types and in BDL-derived LSEC and HSC this was associated with an improvement of its phenotype. Simvastatin (free or encapsulated) caused a significant increase in fenestrae frequency and endothelial porosity. Intravenously administered PM were mainly found in the liver (40% accumulation) both in healthy and cirrhotic rats. Discussion. PM loaded with simvastatin are less toxic than the free drug and are effective improving endothelial and stellate cell phenotype. In vivo administration mainly targets the liver. PM are promising nanocarriers to administer vasoprotective drugs in the context of liver diseases.

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The stabilins are the primary scavenger receptors for Phosphorothioate Antisense Oligonucleotides in Liver Sinusoidal Endothelial Cells

Assoc prof Edward N. Harris1, Colton M. Miller1, Brad Wan2, Hans Gaus2, Punit P. Seth2. Dept of Biochemistry, University of Nebraska1, Lincoln, NE, USA; Dept. of Medicinal Chemistry, Ionis Pharmaceuticals2, Carlsbad, CA, USA. Introduction. The Stabilin-1 (CLEVER-1 and FEEL-1) and Stabilin-2 (HARE and FEEL-2) receptors expressed in liver sinusoidal endothelial cells (SECs) are natural endocytosis and clearance receptors for highly electronegative polymers such as synthetic phosphorothioate-based antisense oligonucleotides (PS-ASOs). The phosphorothioate group replaces the biological phosphodiester in which the non-bridging oxygen in the DNA backbone is substituted with a double-bonded non-bridging sulfur atom. Phosphorothioate chemistry renders the oligo highly resistant to degradation in biological fluids. The phosphorothioate backbone acts as a catalyst for specific mRNA degradation as it bonds with specific RNAs and targets them for RNAase-H degradation while remaining intact. Aims. To determine the role of Stab1 and Stab2 receptors in the overall systemic clearance and activity of PS-ASOs. Methods. A DNA-based 5-10-5 gapmer containing flanking methoxyethyl modifications on the 2’ position of the ribose sugar and a fully phosphorothioate backbone was radiolabelled with 125I on the 5’ end to trace delivery within cells and tissues of whole animals. Activity of the PS-ASO was measured by qPCR against its target sequence, the long non-coding RNA, Malat-1. Results. Injection of 125I-PS-ASO in C57BL/6J mice confirmed that the half-life of this compound is about 9.5 minutes in circulation. Accumulation of most of the material was in liver and within the SECs of liver in contrast to hepatocytes and Kupffer cells. Cell lines expressing either Stabilin-1 or Stabilin-2 rapidly internalized the PS-ASO in a clathrin dependent mechanism. Specific activity for both Stabilins is high as assessed by competitor ASOs that differed in both backbone chemistry and ribose modifications. Binding affinity to the ecto-domain of the small isoform of Stabilin-2 was assessed using three different methods resulting in low nanomolar affinity for 5-10-5 gapmer. Cells/tissues expressing either Stab1 or Stab2 had higher knockdown of Malat-1 expression suggesting endosome escape. Discussion. The Stabilin receptors not only clear the PS-ASOs from circulation, but tissues that express these receptors exhibit higher activation of the PS-ASO such as gene knockdown.

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128

The liver in health and disease: What the intravital microscope reveals

Antony M Wheatley Department of Physiology, School of Medicine, National University of Ireland, Galway, Ireland Changes to the hepatic microcirculation occur in response to liver injury and disease and during liver regeneration follow surgical reduction or transplantation. One method by which the microcirculation may be interrogated is intravital microscopy (IVM), which facilitates the live imaging of cells within their natural environment. IVM can be performed using several light microscopy techniques including widefield fluorescence, confocal and multiphoton microscopy. If tissue penetration greater than 50-100 m is desired, multiphoton microscopy is required. Given the structure of the rodent liver, widefield intravital fluorescence microscopy (IVFM), in combination with the application of fluorophores, allows for the interrogation of the hepatic microcirculation. FITC labelled dextran or erythrocytes may be used to assess intrahepatic blood flow; rhodamine6G to visualize rolling and adhering white blood cells and the nuclear stain Hoescht 33362 to identify normal and apoptotic hepatocytes (Mabuchi et al, 2009; Marlini et al, 2016). Vitamin-A related autofluorescence may be used to identify hepatic stellate cells in the hepatic microcirculation. In the current study, we will look at the impact on the hepatic microcirculation of (i) acute ischaemia/reperfusion (ii) LPS/GalN-induced in normal and stress-sensitive WKY rats (iii) changes that occur to the blood flow in the hepatic microcirculation during liver regeneration following partial hepatectomy and the mystery of the disappearing hepatic arteries. Finally, we will concentrate on (4) bile duct ligation (BDL)-induced liver cirrhosis. Using standard haemodynamic measurements, BDL is shown to be associated with a progressive increase in portal pressure (PP), portal venous blood flow (PVBF) and portal systemic shunting (±65% at 3 weeks), a reduction in hepatic arterial resistance and a 5 fold rise in hepatic arterial blood flow (HABF). Within the hepatic microcirculation, BDL is associated with a progressive fall in sinusoidal diameter, a rise in sinusoidal velocity and reduction in functional sinusoidal density. From our results we conclude that the important changes that occur to the hepatic microcirculation under physiological circumstances (liver regeneration), following acute liver injury and in chronic liver disease may be assessed by IVFM. MABUCHI A, WAKE K, MARLINI M, WATANABE H, WHEATLEY AM (2009). Protection by glycyrrhizin against warm ischemia-reperfusion induced cellular injury and derangement of the microcirculatory blood flow in the rat liver Microcirculation 16, 364-376. MARLINI M, MABUCHI A, MALLARD BL, HAIRULHISYAM N, AKASHI-TAKAMURA A, HARPER JL, WHEATLEY AM (2016). Delayed liver regeneration in C3H/HeJ mice: Possible involvement of haemodynamic and structural changes in the hepatic microcirculation. Experimental Physiology 101, 1492-1505.

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129

Exacerbation of alcoholic liver injury in obese KK-Ay mice involves alteration in small intestinal microbiota signature

Kenichi Ikejima1 Ryuta Kitagawa1, Maiko Suzuki1, Kazuyoshi Kon1, Kyoko Fukuhara1, Akira Uchiyama1 Shunhei Yamashina1, Kyoko Kuwahara-Arai2, Teruo Kirikae2. Department of Gastroenterology1, and Department of Microbiology2, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Introduction. Excessive alcohol consumption is often seen in patients with metabolic syndrome (MetS), and exacerbation of alcoholic hepatitis comorbid MetS is an emerging clinical problem. Gut-derived bacterial products most likely play a profound role in the pathogenesis of both alcoholic liver disease and non-alcoholic steatohepatitis (NASH). Aims. Here, we investigated the alteration of small intestinal microbiota profile following chronic EtOH feeding in obese KK-Ay mice, and the effect of rifaximin (RFX) on liver injury following chronic/binge ethanol (EtOH) administration. Methods. Female, 8-week-old KK-Ay mice were fed Lieber–DeCarli diet (5%EtOH) for 10 days, following a single EtOH gavage (4 g/kg BW). Some mice were given oral administration of RFX (0.1 g/L) in liquid diet. Some mice were anesthetized without binge; small intestinal contents were collected. The net amount of intestinal microbiota was quantified using aerobic and anaerobic conventional culturing techniques, and qualitatively evaluation analysed by 16S rRNA sequencing. Results. The treatment with RFX significantly prevented hepatic steatosis and increase of oxidative stress and inflammatory cytokines in KK-Ay mice given EtOH-feeding/binge. Overexpression of hepatic mRNA levels for cell differentiation (CD)-14 and toll-like receptor 2 (TLR2) following EtOH-feeding/binge was also significantly prevented by RFX. The net Amount of small intestinal bacteria was significantly increased after chronic EtOH feeding as compared to controls; RFX had no effect on the net amount of viable bacterial cells increased by chronic EtOH feeding. In the profile of small intestinal microbiota in the order level, EtOH-feeding dramatically increased the relative abundance of the Erysipelotrichales. RFX drastically reversed the Erysipelotrichales and enriched the Bacteroidales. Discussion. Chronic ethanol administration dramatically changes microbiota signature in small intestine, and RFX-prevents alcoholic liver injury through modulation of small intestinal microbiota/innate immune responses in the liver.

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130

Early hepatic genes expression and signalling pathways in liver regeneration after partial hepatectomy in germ-free mice

Ms Nur Fatin Aqilah Raman, Raman NF Aqilah1, Ngatiman M Hairulhisyam1,2, Manzor N Fariha1, Beth L Mallard2, Fergus Shanahan3, Antony M Wheatley2, Muhamad Marlini1,2. Faculty of Medicine and Health Sciences, USIM1, KL, Malaysia; Department of Physiology, NUIG Galway2, Galway, Ireland; Alimentary Pharmabiotic Centre, UCC3, Cork, Ireland. Introduction. Redundancy of signalling pathways, including immune, proliferative and metabolic pathways exists in liver regeneration. Therefore, loss of an individual gene leads to a delay but not complete inhibition of the regenerative process. To-date, it is believed that intestinal microbiota is involved in liver regeneration as liver regeneration after partial hepatectomy (PH) is delayed in germ-free (GF) mice and antibiotic treated rats. Aim. To investigate the role of the intestinal microbiota in liver regeneration by examining early hepatic genes and signalling pathways following PH in GF mice. Methods. Male, 8-10 weeks old, wild type (WT) (C57BL/6) and GF mice (n=3) were used in this study. Mice underwent PH and liver tissue was collected at 3 hours after PH. Total RNA was extracted for mRNA sequencing. Significantly up- and down-regulated genes in GF mice were subjected to KEGG pathway enrichment analysis using DAVID (p ≤ 0.05). Results. Decreased liver weight-to-body weight ratio was observed in GF mice as compared to WT. 1992 upregulated and 1453 downregulated genes were identified in GF mice as compared to WT. From these up- and down-regulated genes, 18 and 32 KEGG pathways were enriched (p ≤ 0.01). The top enriched up-regulated pathways were metabolic pathways, retinol metabolism and PPAR signalling pathways. The most enriched pathways for down-regulated genes include toll-like receptors, NF-kappa B and TNF signalling pathways. Gene ontology (GO) associated with liver regeneration was overrepresented within the differentially expressed genes. Discussion/Conclusion. Results of the study indicate that; i) decreased expression of immune-pathway-associated hepatic genes leads to a delayed regenerative response in GF mice. ii) Since GF mice lack gut microbiota-associated LPS, disruption of the immune-associated initiation TLR signalling in the regenerative process would appear to occur iii) increased expression of metabolic-pathway-associated hepatic genes in GF mice suggests that this pathway is a dominant regulator in liver regeneration when the LPS/TLR4 signalling pathway is absent. Taken together, our results indicate that the intestinal microbiota is crucial in liver regeneration.

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132

Dietary imbalance of branched amino acids impact fenestrations in liver sinusoidal endothelial cells

Sun Woo Sophie Kang1, David G. Le Couteur1,2 and Victoria C. Cogger1,2. Aging and Alzheimer’s Institute, ANZAC Research Institute1, Concord, Australia. Charles Perkins Centre, University of Sydney2, Sydney, Australia.

It has been previously shown that aging and diets of different macronutrient concentrations can cause defenestration of liver sinusoidal endothelial cells (LSECs) and that defenestrations are associated with impaired insulin transfer and hepatic insulin resistance (1). Among amino acids, branched amino acids (BCAAs) are known to influence insulin, insulin-like growth factor 1 (IGF1) and mammalian target of rapamycin (mTOR)—key pathways linking nutrition with metabolic syndromes and aging. Therefore, in this study we determined how diets differing in concentrations of BCAAs impact LSEC fenestrations in mice using scanning electron microscopy. LSEC morphology was analyzed in C57BL/6J mice that were fed diets with different concentrations of BCAAs compared to a standard chow diet for 15 months. These diets were: BCAA200, which had 200 % the amount of BCAAs compared to chow; BCAA50, which had 50 % of the BCAAs compared to chow; and BCAA20, which had 20 % of the BCAAS compared to standard chow. A control group, BCAA100, which was fed standard chow was also included. Half of the mice were given ad libitum access to these diets and half were maintained on a strict 80 % caloric restriction diet of these diets. Preliminary results indicate that there was a significant decrease in the frequency of fenestrations with all BCAA diets compared to the control but no significant changes to the porosity and diameter compared to the control diet (BCAA100). Further analysis indicated that caloric restriction of all diets led to increased fenestration diameter, frequency and LSEC porosity. Overall the results provide preliminary evidence that long term dietary amino acid imbalance altered the morphology of LSECs, but this may be reversed by calorie restriction. Cogger VC, Mohamad M, Solon-Biet SM, Senior AM, Warren A, O'Reilly JN, Tung BT, Svistounov D, McMahon AC, Fraser R, Raubenheimer D, Holmes AJ, Simpson SJ, and Le Couteur DG. Dietary macronutrients and the aging liver sinusoidal endothelial cell. American journal of physiology Heart and circulatory physiology 310: H1064-1070, 2016.

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133

Cytoglobin suppresses liver fibrosis and cancer in mice by regulating oxidative stress

Le Thi Thanh Thuy¹, Ninh Quoc Dat¹, Hoang Hai¹, Dinh Viet Hoang¹, Katsutoshi Yoishizato² and Norifumi Kawada1*. 1Dept of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan; 2PhoenixBio Co. Ltd., Hiroshima, Japan Introduction. Cytoglobin (CYGB) is a gas-binding protein presenting in hepatic stellate cell (HSCs) and functions as anti-oxidant. We, for the first time, generated Cygb-transgenic (TG) mice to access its role in fibrosis and cancer. Aim. We assessed the effects of CYGB on bile duct ligation (BDL) induced biliary fibrosis, thioacetamide (TAA)-induced fibrosis, and diethylnitrosamine (DEN) induced liver cancer. Method. TG mice were created using native Cygb promoter to drive expression of exogenous Cygb and mCherry reporter allowing to tracing the cells. Cygb transgenic (TG) and Cygb knockout (KO) mice were sacrificed 1, 2, and 3 weeks after BDL, or 10 weeks after TAA injection, or 12 months after injection of DEN at 6 mg/kg BW at 15 days old. Results. CYGB and mCherry co-localized in cytoplasm of pericytes of all organs including HSCs in the livers and ten-fold increase compared to endogenous Cygb level. (1) In BDL model, compared to WT, fibrosis was robustly developed in KO mice indicated by markedly increased expression of α-SMA, collagen 1a1, and Sirius-red positive area. TG mice showed suppression of these factors up to 55% compared to WT mice. Oxidative stress were markedly increased in KO but inhibited in TG livers. Of note, silencing Cygb or ROS inducer in primary HSCs isolated from WT mice promoted their activation and collagen production. Interestingly, these effects were inhibited in HSCs isolated from TG mice. Microarray analysis from 1 week of BDL in all WT, KO, and TG livers indicated that CYGB regulate oxidative stress pathway including Nox-1, antioxidant protein 1, N-acetyltransferase 8, and myeloperoxidase. (2) In TAA model, TG mice significantly ameliorated liver inflammation and fibrosis compared to WT ones. (3) In DEN model, number and maximum size of liver tumors were significantly regressed in TG mice compared to WT ones. Liver fibrosis indicated by αSMA, desmin, oxidative stress shown by 4-HNE, and inflammation as shown by CD68 staining showed down regulation in TG liver. In contrast, anti-oxidant factor NRF-2 was significantly increased in TG mice compared to WT. Discussion. Cell specific expression of Cygb may regulate HSC activation and supressed the liver inflammation, fibrosis and cancer via ROS scavenger function.

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134

Transcriptomic signature of LSECs during the progression of cirrhosis: from inflammation to cancer development

Anabel Fernández-Iglesias1, Nicolò Manicardi1, Laia Abad1, Juan José Lozano1, Jordi Gracia-Sancho1,2. Liver Vascular Biology Research Group, IDIBAPS, CIBERehd1, Barcelona, Spain; University of Bern2, Switzerland. Introduction. In advanced chronic liver disease (CLD), liver sinusoidal endothelial cells (LSECs) become profoundly deregulated showing alterations in their phenotype and function. We hypothesize that a deep characterization of their transcriptomic profile during the progression of CLD would help to better understand the process of LSEC capillarization and to reveal therapeutic targets specific for each stage of the disease. Aims. We aimed at analysing the transcriptomic signature of primary LSECs during the progression of cirrhosis. Methods. Highly pure and viable LSECs were isolated from healthy rats and from rats with liver injury due to CCl4 inhalation at 3 stages of the disease: after 2 weeks of CCl4 (acute liver injury), after 6 weeks (fibrosis), and after 14 weeks (cirrhosis). Total RNA libraries were constructed following TruSeq Stranded mRNA Library Prep Guide and sequenced in an Illumina platform HiSeq2500 where reads were mapped against Rattus norvegicus genome (Rnor_6.0). Quantification of genes was performed with RSEM using ensembl release 95. Functional analysis of up- and down-regulated genes with fold-change>2 comparing healthy and injured LSECs was executed with Integrity Pathway Analysis. Canonical pathways and functions annotation with p<0.05 were considered. Results. The transcriptomic analyses of LSECs revealed that 7349 genes were deregulated in cells from acute injured livers, 6989 genes in liver fibrosis and 4069 genes in liver cirrhosis. Integrity pathway analyses showed that in the first stage of the disease LSECs de-differentiation mainly focuses on inflammatory (46% of de-regulated pathways) and cell-to-cell interaction (52%) processes. Fibrotic and cirrhotic LSECs exhibit a more complex transcriptomic signature sharing major deregulations in cellular organization and morphology (15-20% of total deregulated pathways), cellular metabolism (14%) and cell death (13%). Nevertheless, fibrotic LSECs presented a marked enrichment in pathways implicated in cell proliferation and development, including angiogenesis and vascular proliferation, while cirrhotic LSECs had a notorious increase in pathways implicated in cell transformation & invasion, and tumour development. Discussion. Whole transcriptome sequencing of LSECs during the progression of chronic liver disease revealed particular chronological changes in the pathobiological processes implicated in endothelial dedifferentiation. This new knowledge defines temporary-specific therapeutic targets for the amelioration of LSECs and chronic liver disease.