Delineating the heterogeneity of senescence-induced-functional alterations in hepatocytes.

BACKGROUND AND AIM: Cellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory cytokines. This 'zombie-like' state perpetuates harmful effects on tissues and holds potential implications for liver disease progression. Remarkably, senescence exhibits heterogeneity, stemming from two crucial factors: the inducing stressor and the cell type. As such, our present study endeavors to characterize stressor-specific changes in senescence phenotype, its related molecular patterns, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs). METHODS: PMH, isolated by collagenase-perfused mouse liver (C57B6/J; 18-23 weeks), were cultured overnight in William's E-medium supplemented with 2% FBS, L-glutamine, and hepatocyte growth supplements. HepOrgs were developed by culturing cells in a 3D matrix for two weeks. The senescence was induced by DNA damage (doxorubicin, cisplatin, and etoposide), oxidative stress (H2O2, and ethanol), and telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyl transferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine and hydroxyurea). SA-ß galactosidase activity, immunofluorescence, immunoblotting, and senescence-associated secretory phenotype (SASP), and cellular bioenergetics were used to assess the senescence phenotype. RESULTS: Each senescence inducer triggers a unique combination of senescence markers in hepatocytes. All senescence inducers, except hydroxyurea and ethanol, increased SA-ß galactosidase activity, the most commonly used marker for cellular senescence. Among the SASP factors, CCL2 and IL-10 were consistently upregulated, while Plasminogen activator inhibitor-1 exhibited global downregulation across all modes of senescence. Notably, DNA damage response was activated by DNA damage inducers. Cell cycle markers were most significantly reduced by doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence shifted cellular bioenergetics capacity from glycolysis to oxidative phosphorylation. In HepOrgs exposed to senescence inducers, there was a notable increase in γH2A.X, p53, and p21 levels. Interestingly, while showing a similar trend, SASP gene expression in HepOrgs was significantly higher compared to PMH, demonstrating a several-fold increase. CONCLUSION: In our study, we demonstrated that each senescence inducer activates a unique combination of senescence markers in PMH. Doxorubicin demonstrated the highest efficacy in inducing senescence, followed by cisplatin and H2O2, with no impact on apoptosis. Each inducer prompted DNA damage response and mitochondrial dysfunction, independent of MAPK/AKT.

Congenital Afibrinogenemia With Facial Haematoma.

Congenital afibrinogenemia is a rare inherited blood disorder characterized by a deficiency of fibrinogen, leading to abnormal blood clotting. It is caused by mutations in fibrinogen genes and results in a propensity for bleeding. We present the case of a one-year-old male child with congenital afibrinogenemia who developed a left-sided facial haematoma following a fall from a walker. The child had a history of active bleeding during cannulation and had not undergone circumcision due to the risk of bleeding. This case highlights the need for timely diagnosis and appropriate management of rare bleeding disorders such as congenital afibrinogenemia. Collaboration between different specialties, including haematology and genetic counseling, is crucial for comprehensive care. The rarity of the condition underscores the importance of raising awareness among healthcare professionals. Genetic counseling and family studies are essential for assessing genetic implications and guiding decision-making. Further advancements in diagnostic tests and replacement therapy are needed to improve the management of patients with afibrinogenemia, particularly in regions with a high prevalence of consanguineous marriages.

Single-cell landscape of immune cells in human livers affected by HBV-related cirrhosis.

Background & Aims: HBV infection is one of the leading causes of liver cirrhosis. However, the immune microenvironment in patients with HBV cirrhosis remains elusive. Methods: Single-cell RNA sequencing was used to analyse the transcriptomes of 76,210 immune cells in the livers of six healthy individuals and in five patients with HBV cirrhosis. Results: Patients with HBV cirrhosis have a unique immune ecosystem characterised by an accumulation of macrophage-CD9/IL18, macrophage-C1QA, NK Cell-JUNB, CD4+ T cell-IL7R, and a loss of B cell-IGLC1 clusters. Furthermore, our analysis predicted enhanced cell communication between myeloid cells and all immune cells in patients with HBV-related cirrhosis. Pseudo-time analysis of myeloid cells, natural killer (NK) cells, and B cells demonstrated a significant accumulation of mature cells and a depletion of naive cells in HBV cirrhosis. In addition, we observed an increase in antigen processing and presentation capacities in myeloid cells in patients with HBV cirrhosis, whereas NK cell-mediated cytotoxicity was substantially reduced. Conclusions: Our results provide valuable insight into the immune landscape of HBV cirrhosis, suggesting that HBV cirrhosis is associated with the accumulation of activated myeloid cells and impaired cytotoxicity in NK cells. Impact and implications: The absence of single-cell transcriptome profiling of immune cells in HBV cirrhosis hinders our understanding of the underlying mechanisms driving disease progression. To address this knowledge gap, our study unveils a distinctive immune ecosystem in HBV cirrhosis and represents a crucial advancement in elucidating the impact of the immune milieu on the development of this condition. These findings constitute significant strides towards the identification of more effective therapeutic approaches for HBV cirrhosis and are relevant for healthcare professionals, researchers, and pharmaceutical developers dedicated to combating this disease.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension.

Intestinal microbiota can modulate portal hypertension through the regulation of the intestinal vasculature. We have recently demonstrated that bacterial antigens activate Paneth cells (PCs) to secrete products that regulate angiogenesis and portal hypertension. In the present work we hypothesized that Paneth cells regulate the development of lymphatic vessels under the control of intestinal microbiota during experimental portal hypertension. We used a mouse model of inducible PCs depletion (Math1Lox/LoxVilCreERT2) and performed partial portal vein ligation (PPVL) to induce portal hypertension. After 14 days, we performed mRNA sequencing and evaluated the expression of specific lymphangiogenic genes in small intestinal tissue. Intestinal and mesenteric lymphatic vessels proliferation was assessed by immunohistochemistry. Intestinal organoids with or without PCs were exposed to pathogen-associated molecular patterns, and conditioned media (CM) was used to stimulate human lymphatic endothelial cells (LECs). The lymphangiogenic activity of stimulated LECs was assessed by tube formation and wound healing assays. Secretome analysis of CM was performed using label-free proteomics quantification methods. Intestinal immune cell infiltration was evaluated by immunohistochemistry. We observed that the intestinal gene expression pattern was altered by the absence of PCs only in portal hypertensive mice. We found a decreased expression of specific lymphangiogenic genes in the absence of PCs during portal hypertension, resulting in a reduced proliferation of intestinal and mesenteric lymphatic vessels as compared to controls. In vitro analyses demonstrated that lymphatic tube formation and endothelial wound healing responses were reduced significantly in LECs treated with CM from organoids without PCs. Secretome analyses of CM revealed that PCs secrete proteins that are involved in lipid metabolism, cell growth and proliferation. Additionally, intestinal macrophages infiltrated the ileal mucosa and submucosa of mice with and without Paneth cells in response to portal hypertension. Our results suggest that intestinal microbiota signals stimulate Paneth cells to secrete factors that modulate the intestinal and mesenteric lymphatic vessels network during experimental portal hypertension.

Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model. RESULTS: In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration. CONCLUSION: The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF. LAY SUMMARY: Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

Prognostic Role of Liver Biopsy in Patients With Severe Indeterminate Acute Hepatitis.

BACKGROUND AND AIMS: Severe indeterminate acute hepatitis (sIAH) is a poorly understood rare disease with no specific therapy. This study aims to define the clinicopathological characteristics of sIAH and the role of liver biopsy in determining prognosis. METHODS: Patients with sIAH admitted to a single center between 2010 and 2019 were included. Histopathological patterns of liver biopsies were reviewed by 2 histopathologists, and key findings further were specified by multiplex immunofluorescence. Patients that died or underwent liver transplantation were analyzed as nonsurvivors. RESULTS: Of 294 patients with acute hepatitis, 43 with sIAH were included. Seventeen (39.5%) underwent liver transplantation and 7 (16.2%) died within 3 months. Multilobular necrosis was the predominant histopathological feature, being significantly more frequent in nonsurvivors (62.5% vs 21.1%; P = .016). Necrotic areas showed low HNF4α and Ki67 expression but high expression of CK19 and cell death markers identifying areas of severe tissue injury and inadequate regenerative response. Patients with multilobular necrosis had higher international normalized ratio, Model for End-Stage Liver Disease, and Model for End-Stage Liver Disease-Sodium scores compared with those without (P values for all markers <.05). Multivariate Cox analysis revealed that multilobular necrosis (hazard ratio, 3.675; 95% confidence interval, 1.322-10.211) and lower body mass index (hazard ratio, 0.916; 95% confidence interval, 0.848-0.991) independently predicted death or transplantation. CONCLUSIONS: The results of this study provide novel insights into the important role of liver biopsy in sIAH patients, suggesting that the presence of multilobular necrosis is an early indicator of poor prognosis.

Presence of multilobular necrosis on liver biopsy identifies corticosteroid responsiveness in acute indeterminate hepatitis.

BACKGROUND AND AIMS: Treatment of patients with severe indeterminate hepatitis (IAH) is an unmet need. Corticosteroids are often used in the management of these patients but criteria for the selection of patients for this intervention are arbitrary. The aims of this study were to analyse the clinical and pathological features of patients with IAH to define predictors of corticosteroid responsiveness. METHODS: This study included consecutive patients with acute indeterminate hepatitis admitted to a single hospital and underwent a liver biopsy. The clinical manifestation and histopathological features of steroid and non-steroid groups were compared and their relationship with corticosteroids response was evaluated. RESULTS: Forty-eight patients were included, 24 (50%) recovered and the other half underwent liver transplantation or died within 3-months. Of the 48 cases, 24 received corticosteroids (initial dose of 45 ± 12 mg prednisolone). Corticosteroids were initiated 2.7 ± 3.8 days after admission. Liver biopsy was performed 2-days (median, IQR 1-3) after admission. Fifteen (62.5%) patients receiving corticosteroids survived without transplantation compared with 9 (37.5%) that did not receive steroids (P = .149). In those with multilobular necrosis, 50% reduction in the death/transplantation rate was observed after steroid treatment (P = .018). In patients without multilobular necrosis and with or without perivenulitis, corticosteroids did not impact the outcome. Response to corticosteroids was independent of the MELD score. CONCLUSIONS: The presence of multilobular necrosis on liver biopsy helps identify a subgroup of IAH cases who may benefit from the administration of corticosteroids.

Intestinal microbiota drives cholestasis-induced specific hepatic gene expression patterns.

Intestinal microbiota regulates multiple host metabolic and immunological processes. Consequently, any difference in its qualitative and quantitative composition is susceptible to exert significant effects, in particular along the gut-liver axis. Indeed, recent findings suggest that such changes modulate the severity and the evolution of a wide spectrum of hepatobiliary disorders. However, the mechanisms linking intestinal microbiota and the pathogenesis of liver disease remain largely unknown. In this work, we investigated how a distinct composition of the intestinal microbiota, in comparison with germ-free conditions, may lead to different outcomes in an experimental model of acute cholestasis. Acute cholestasis was induced in germ-free (GF) and altered Schaedler's flora (ASF) colonized mice by common bile duct ligation (BDL). Studies were performed 5 days after BDL and hepatic histology, gene expression, inflammation, lipids metabolism, and mitochondrial functioning were evaluated in normal and cholestatic mice. Differences in plasma concentration of bile acids (BA) were evaluated by UHPLC-HRMS. The absence of intestinal microbiota was associated with significant aggravation of hepatic bile infarcts after BDL. At baseline, we found the absence of gut microbiota induced altered expression of genes involved in the metabolism of fatty and amino acids. In contrast, acute cholestasis induced altered expression of genes associated with extracellular matrix, cell cycle, autophagy, activation of MAPK, inflammation, metabolism of lipids, and mitochondrial functioning pathways. Ductular reactions, cell proliferation, deposition of collagen 1 and autophagy were increased in the presence of microbiota after BDL whereas GF mice were more susceptible to hepatic inflammation as evidenced by increased gene expression levels of osteopontin, interleukin (IL)-1ß and activation of the ERK/MAPK pathway as compared to ASF colonized mice. Additonally, we found that the presence of microbiota provided partial protection to the mitochondrial functioning and impairment in the fatty acid metabolism after BDL. The concentration of the majority of BA markedly increased after BDL in both groups without remarkable differences according to the hygiene status of the mice. In conclusion, acute cholestasis induced more severe liver injury in GF mice compared to mice with limited intestinal bacterial colonization. This protective effect was associated with different hepatic gene expression profiles mostly related to tissue repair, metabolic and immune functions. Our findings suggest that microbial-induced differences may impact the course of cholestasis and modulate liver injury, offering a background for novel therapies based on the modulation of the intestinal microbiota.

Linear and branched ß-Glucans degrading enzymes from versatile Bacteroides uniformis JCM 13288T and their roles in cooperation with gut bacteria.

ß-glucans are the dietary nutrients present in oats, barley, algae, and mushrooms. The macromolecules are well known for their immune-modulatory activity; however, how the human gut bacteria digest them is vaguely understood. In this study, Bacteroides uniformis JCM 13288 T was found to grow on laminarin, pustulan, and porphyran. We sequenced the genome of the strain, which was about 5.05 megabase pairs and contained 4868 protein-coding genes. On the basis of growth patterns of the bacterium, two putative polysaccharide utilization loci for ß-glucans were identified from the genome, and associated four putative genes were cloned, expressed, purified, and characterized. Three glycoside hydrolases (GHs) that were endo-acting enzymes (BuGH16, BuGH30, and BuGH158), and one which was an exo-acting (BuGH3) enzyme. The BuGH3, BuGH16, and BuGH158 can cleave linear exo/endo- ß- 1-3 linkages while BuGH30 can digest endo- ß- 1-6 linkages. BuGH30 and BuGH158 were further explored for their roles in digesting ß- glucans and generation of oligosaccharides, respectively. The BuGH30 predominately found to cleave long chain ß- 1-6 linked glucans, and obtained final product was gentiobiose. The BuGH158 used for producing oligosaccharides varying from degree of polymerization 2 to 7 from soluble curdlan. We demonstrated that these oligosaccharides can be utilized by gut bacteria, which either did not grow or poorly grew on laminarin. Thus, B. uniformis JCM 13288 T is not only capable of utilizing ß-glucans but also shares these glycans with human gut bacteria for potentially maintaining the gut microbial homeostasis.

Cirrhotic Endothelial Progenitor Cells Enhance Liver Angiogenesis and Fibrosis and Aggravate Portal Hypertension in Bile Duct-Ligated Cirrhotic Rats.

BACKGROUND: Circulating cirrhotic endothelial progenitor cells (EPC) interact with both liver sinusoidal endothelial cells (LSEC) and hepatic stellate cells (HSC) and promote angiogenesis in vitro. This study evaluated the effect of cirrhotic and control EPCs on hepatic angiogenesis, microcirculation, and fibrosis in vivo in rat models of cirrhosis. METHODOLOGY: Animal models of cirrhosis were prepared by bile duct ligation (BDL). Circulating EPCs isolated from healthy human and cirrhotic blood were characterized by flow cytometry, cultured and administered through the tail vein in BDL rats after 2 weeks of ligation. The cells were given thrice a week for 2 weeks. The untreated group of BDL rats received only saline. Fibrosis was evaluated by Masson's trichrome staining. Dedifferentiated LSECs were identified by the expression of CD31, and activated HSCs were marked as alpha-SMA-positive cells and were studied by immunohistochemistry and western blotting in saline-, healthy EPC-, and cirrhotic EPC-treated rats. In vivo, hepatic and systemic hemodynamic parameters were evaluated. Liver functions were evaluated. RESULTS: In comparison to controls, BDL rats revealed an increase of fibrosis and angiogenesis. Among the treated rats, cirrhotic EPC-treated rats had increased fibrosis grade as compared to healthy EPC-treated and saline-treated rats. There was an increase of both fibrosis and angiogenesis markers, alpha-SMA and CD31 in cirrhotic EPC-treated rats as compared to healthy EPC-treated and saline-treated rats in immunohistochemistry and western blot studies. Cirrhotic EPC-treated BDL rats had high portal pressure and portal blood flow with significantly elevated hepatic vascular resistance in comparison with healthy EPC- and saline-treated BDL animals, without significant differences in mean arterial pressure. Cirrhotic EPC-treated BDL rats also showed a substantial increase in the hepatic expression of angiogenic receptors, VEGFR2 and CXCR4 in comparison with saline-treated rats. CONCLUSION: The study suggests that transplantation of cirrhotic EPCs enhances LSEC differentiation and angiogenesis, activates HSCs and worsens fibrosis, thus resulting in hepatic hemodynamic derangements in BDL-induced cirrhosis.

Paneth cells promote angiogenesis and regulate portal hypertension in response to microbial signals.

BACKGROUND & AIMS: Paneth cells (PCs) synthesize and secrete antimicrobial peptides that are key mediators of host-microbe interactions, establishing a balance between intestinal microflora and enteric pathogens. We observed that their number increases in experimental portal hypertension and aimed to investigate the mechanisms by which these cells can contribute to the regulation of portal pressure. METHODS: We first treated Math1Lox/LoxVilcreERT2 mice with tamoxifen to induce the complete depletion of intestinal PCs. Subsequently, we performed partial portal vein or bile duct ligation. We then studied the effects of these interventions on hemodynamic parameters, proliferation of blood vessels and the expression of genes regulating angiogenesis. Intestinal organoids were cultured and exposed to different microbial products to study the composition of their secreted products (by proteomics) and their effects on the proliferation and tube formation of endothelial cells (ECs). In vivo confocal laser endomicroscopy was used to confirm the findings on blood vessel proliferation. RESULTS: Portal hypertension was significantly attenuated in PC-depleted mice compared to control mice and was associated with a decrease in portosystemic shunts. Depletion of PCs also resulted in a significantly decreased density of blood vessels in the intestinal wall and mesentery. Furthermore, we observed reduced expression of intestinal genes regulating angiogenesis in Paneth cell depleted mice using arrays and next generation sequencing. Tube formation and wound healing responses were significantly decreased in ECs treated with conditioned media from PC-depleted intestinal organoids exposed to intestinal microbiota-derived products. Proteomic analysis of conditioned media in the presence of PCs revealed an increase in factors regulating angiogenesis and additional metabolic processes. In vivo endomicroscopy showed decreased vascular proliferation in the absence of PCs. CONCLUSIONS: These results suggest that in response to intestinal flora and microbiota-derived factors, PCs secrete not only antimicrobial peptides, but also pro-angiogenic signaling molecules, thereby promoting intestinal and mesenteric angiogenesis and regulating portal hypertension. LAY SUMMARY: Paneth cells are present in the lining of the small intestine. They prevent the passage of bacteria from the intestine into the blood circulation by secreting substances to fight bacteria. In this paper, we discovered that these substances not only act against bacteria, but also increase the quantity of blood vessels in the intestine and blood pressure in the portal vein. This is important, because high blood pressure in the portal vein may result in several complications which could be targeted with novel approaches.

Isoproterenol Disrupts Intestinal Barriers Activating Gut-Liver-Axis: Effects on Intestinal Mucus and Vascular Barrier as Entry Sites.

BACKGROUND: The gut-liver-axis presents the pathophysiological hallmark for multiple liver diseases and has been proposed to be modulated during stress and shock. Access to the gut-liver-axis needs crossing of the mucus and gut-vascular barrier. The role of ß-adrenoreceptor-activation for both barriers has not been defined and is characterized here. METHODS: Splanchnic ß-adrenergic stimulation was achieved by chronic intraperitoneal application of isoproterenol via alzet-pump in vivo. The intestinal permeability and gut-vascular barrier function was assessed in ileal loop experiments. The extravasation of predefined sizes of fluorescence isothiocyanate (FITC)-dextran molecules in ileal microcirculation was evaluated by intravital confocal laser endomicroscopy in vivo. Mucus parameters thickness, goblet cell count and mucin-expression were assessed by stereomicroscopy, immunostaining and RNA-sequencing respectively. Ileal lamina propria (LP) as well as mesenteric lymph node mononuclear cells was assessed by FACS. RESULTS: Healthy mice lack translocation of 4 kDa-FITC-dextran from the small intestine to the liver, whereas isoproterenol-treated mice demonstrate pathological translocation (PBT). Mucus layer is reduced in thickness with loss of goblet-cells and mucin-2-staining and -expression in isoproterenol-treated animals under standardized gnotobiotic conditions. Isoproterenol disrupts the gut vascular barrier displaying Ileal extravasation of large-sized 70- and 150 kDa-FITC-dextran. This pathological endothelial permeability and accessibility induced by isoproterenol associates with an augmented expression of plasmalemmal-vesicle-associated-protein-1 in intestinal vessel. Ileal LP after isoproterenol treatment contains more CD11c+-dendritic cells (DC) with increased appearance of CCR7+ DC in mesenteric lymph nodes. CONCLUSIONS: Isoproterenol impairs the intestinal muco-epithelial and endothelial-vascular barrier promoting PBT to the liver. This barrier dysfunction on multiple levels potentially can contribute to liver injury induced by catecholamines during states of increased ß-adrenergic drive.

Increased Expression of RUNX1 in Liver Correlates with NASH Activity Score in Patients with Non-Alcoholic Steatohepatitis (NASH).

Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF- significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH.

Attenuated fibrosis in specific pathogen-free microbiota in experimental cholestasis- and toxin-induced liver injury.

In advanced chronic liver disease (CLD), the translocation of intestinal bacteria and the resultant increase of proinflammatory cytokines in the splanchnic and systemic circulation may contribute to the progression of fibrosis. We therefore speculated that fibrosis and portal hypertension (PHT) would be attenuated in a mouse model of limited intestinal colonization with altered Schaedler flora (ASF) compared to a more complex colonization with specific pathogen-free (SPF) flora. We induced liver fibrosis in ASF and SPF mice by common bile duct ligation (BDL) or by carbon tetrachloride (CCl4) treatment. We then measured portal pressure (PP), portosystemic shunts (PSSs), and harvested tissues for further analyses. There were no differences in PP between sham-treated ASF or SPF mice. After BDL or CCl4 treatment, PP, PSSs, and hepatic collagen deposition increased in both groups. However, the increase in PP and the degree of fibrosis was significantly higher in ASF than SPF mice. Expression of fibrotic markers α-smooth muscle actin, desmin, and platelet-derived growth factor receptor ß were significantly higher in ASF than SPF mice. This was associated with higher activation of hepatic immune cells (macrophages, neutrophils) and decreased expression of the intestinal epithelial tight junction proteins (claudin-1, occludin-1). In 2 models of advanced CLD, SPF mice presented significantly attenuated liver injury, fibrosis, and PHT compared to ASF mice. In contrast to our hypothesis, these findings suggest that a complex intestinal microbiota may play a "hepato-protective" role.-Moghadamrad, S., Hassan, M., McCoy, K. D., Kirundi, J., Kellmann, P., De Gottardi, A. Attenuated fibrosis in specific pathogen-free microbiota in experimental cholestasis- and toxin-induced liver injury.

Endothelial Cell-Derived TGF-ß Promotes Epithelial-Mesenchymal Transition via CD133 in HBx-Infected Hepatoma Cells.

Background: Hepatitis B-X Protein (HBx) encoded in Hepatitis B virus (HBV) is known to play a critical role in development and progression of HBV induced hepatocellular carcinoma (HCC). HBx interacts with and activates various cells in HCC microenvironment to promote tumor initiation, progression and invasion. In this study, we investigated how surrounding stromal cells interact with HBx-infected hepatoma cells by a series of in vitro co-culture studies. Methods: Huh7 hepatoma cells were cultured and transfected with the mammalian expression vector pGFP-HBx. Co-culture assays were performed between HBx-transfected Huh7 cells and conditioned media (CM) from stromal cells [endothelial cell lines (HUVECs) and hepatic stellate cell lines (LX2 cells)]. The effect of these interactions was studied by a series of functional assays like chemotaxis, invasion, and wound healing scratch assays. Also, quantitative real time (RT)-PCRs of the mesenchymal genes was performed in the hepatoma cells with and without the co-cultures. Hep3B cells with an integrated HBV genome were taken as positive controls. Results: HBx-transfected Huh7 cells cultured in presence of CM from HUVECs illustrated enhanced migration and tube formation as compared to HBx-transfected cells cultured alone or co-cultured with LX2 cells. HBx-transfected hepatoma cells incubated with CM from HUVECs also expressed mesenchymal genes including Thy1, CDH2, TGFßR1, VIM, and CD133. ELISAs revealed increased levels of TGF-ß in CM from HUVECs. In comparison to unstimulated HBx-transfected Huh7 cells, TGF-ß stimulated cells displayed increased invasive properties and mesenchymal gene expression. RT-PCR and flow cytometry analysis further demonstrated that incubation with either CM from HUVECs or TGF-ß significantly increased the expression of a stemness marker, CD133 in HBx-infected hepatoma cells. Gene inhibition experiments with CD133 siRNA showed a downregulation of mesenchymal gene expression and properties in TGF-ß induced HBx-infected hepatoma cells as compared to that observed in control siRNA treated cells, indicating CD133 as one of the key molecules affecting epithelial to mesenchymal transition (EMT) in HBx-infected cells. Conclusion: The study indicates that secretory factors like TGF-ß from neighboring endothelial cells may enhance expression of CD133 and impart an aggressive EMT phenotype to HBx-infected hepatoma cells in HBV induced HCC.

Alleviating liver failure conditions using an integrated hybrid cryogel based cellular bioreactor as a bioartificial liver support.

Conventionally, some bioartificial liver devices are used with separate plasmapheresis unit to separate out plasma from whole blood and adsorbent column to detoxify plasma before it passes through a hepatocytes-laden bioreactor. We aim to develop a hybrid bioreactor that integrates the separate modules in one compact design improving the efficacy of the cryogel based bioreactor as a bioartificial liver support. A plasma separation membrane and an activated carbon cloth are placed over a HepG2-loaded cryogel scaffold in a three-chambered bioreactor design. This bioreactor is consequently connected extracorporeally to a rat model of acute liver failure for 3 h and major biochemical parameters studied. Bilirubin and aspartate transaminase showed a percentage decrease of 20-60% in the integrated bioreactor as opposed to 5-15% in the conventional setup. Urea and ammonia levels which showed negligible change in the conventional setup increase (40%) and decrease (18%), respectively in the integrated system. Also, an overall increase of 5% in human albumin in rat plasma indicated bioreactor functionality in terms of synthetic functions. These results were corroborated by offline evaluation of patient plasma. Hence, integrating the plasmapheresis and adsorbent units with the bioreactor module in one compact design improves the efficacy of the bioartificial liver device.