Expression of ECM proteins fibulin-1 and -2 in acute and chronic liver disease and in cultured rat liver cells

Fibulin-2 has previously been considered as a marker to distinguish rat liver myofibroblasts from hepatic stellate cells. The function of other fibulins in acute or chronic liver damage has not yet been investigated. The aim of this study has been to evaluate the expression of fibulin-1 and -2 in models of rat liver injury and in human liver cirrhosis. Their cellular sources have also been investigated. In normal rat liver, fibulin-1 and -2 were both mainly present in the portal field. Fibulin-1-coding transcripts were detected in total RNA of normal rat liver, whereas fibulin-2 mRNA was only detected by sensitive, real-time quantitative polymerase chain reaction. In acute liver injury, the expression of fibulin-1 was significantly increased (17.23-fold after 48 h), whereas that of fibulin-2 was not modified. The expression of both fibulin-1 and -2 was increased in experimental rat liver cirrhosis (19.16- and 26.47-fold, respectively). At the cellular level, fibulin-1 was detectable in hepatocytes, “activated” hepatic stellate cells, and liver myofibroblasts (2.71-, 122.65-, and 469.48-fold over the expression in normal rat liver), whereas fibulin-2 was restricted to liver myofibroblasts and was regulated by transforming growth factor beta-1 (TGF-β1) in 2-day-old hepatocyte cultures and in liver myofibroblasts. Thus, fibulin-1 and -2 respond differentially to single and repeated damaging noxae, and their expression is differently present in liver cells. Expression of the fibulin-2 gene is regulated by TGF-β1 in liver myofibroblasts

Investigation of defect formation and electronic transport in microcrystalline silicon deposited by hot-wire CVD

We have investigated doped and undoped layers of microcrystalline silicon prepared by hot-wire chemical vapour deposition optically, electrically and by means of transmission electron microscopy. Besides needle-like crystals grown perpendicular to the substrate's surface, all of the layers contained a noncrystalline phase with a volume fraction between 4% and 25%. A high oxygen content of several per cent in the porous phase was detected by electron energy loss spectrometry. Deep-level transient spectroscopy of the crystals suggests that the concentration of electrically active defects is less than 1% of the undoped background concentration of typically 10^17 cm -3. Frequency-dependent measurements of the conductance and capacitance perpendicular to the substrate surface showed that a hopping process takes place within the noncrystalline phase parallel to the conduction in the crystals. The parasitic contribution to the electrical circuit arising from the porous phase is believed to be an important loss mechanism in the output of a pin-structured photovoltaic solar cell deposited by hot-wire CVD

Implantation of a colorectal stent as a therapeutic approach in the treatment of esophageal leakage

BACKGROUND: While the mortality of esophageal surgery has decreased due to technological advancements, there is still a complication rate of about 30%. One of the main complications is the anastomotic leakage associated with a significant rate of morbidity and mortality. To close the leakage the efficacy of self-expanding stents (SES) has been shown in different studies. However, the high rate of stent migration limits the use of commercial available stents. In our case we were faced with the problem that the diameter of all available stents was too small to attach tightly to the mucosal wall of the esophagogastric anastomosis. CASE PRESENTATION: We used, for the first time to our knowledge, a metal stent designed for colorectal application in an extensive anastomotic leak after esophageal resection in a patient with an esophageal cancer. After primary surgery with subtotal esohagectomy the anastomotic leak was stented endoscopically with a Polyflex self-expanding covered plastic stent after no response to intensive conventional management. Even though the stent was placed correctly, the diameter of the Polyflex stent was too small to attach onto the wall of the esophagogastric anastomosis. Again surgery was performed with a thoracal resection of the esophageal remnant and a hand made anastomosis. Unfortunately, again an anastomotic leak was detected soon after. To close the leak we decided to use a covered colorectal stent (Hanarostent) with an inner diameter of 30 mm. Sixteen weeks later the stent was extracted and complete mucosal healing of the esophageal leak was observed. CONCLUSION: The stent implantation with a large wide diameter offers a good chance to close more extensive leaks and prevent stent migration

An Immune Gene Expression Signature Associated With Development of Human Hepatocellular Carcinoma Identifies Mice That Respond to Chemopreventive Agents

Program (HEPCAR, reference no. 667273-2); US Department of Defense(CA150272P3); an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER,reference no. C9380/A26813), NCI Cancer Center Support Grant, National Cancer Institute; Tisch Cancer Institute (P30-CA196521); Samuel Waxman Cancer Research Foundation; Spanish National Health Institute (SAF2016-76390); and the Generalitat de Catalunya/AGAUR (SGR-1358). Agrin Moeini is supported by Spanish National Health Institute. Sara Torrecilla and Judit Peix are funded by Centro de Investigación Biomedica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd-ISCIII). Carla Montironi is a recipient of Josep Font grant. Carmen Andreu-Oller is supported by "la Caixa" INPhINIT Fellowship Grant (LCF/BQ/IN17/11620024). Roser Pinyol is supported by HEPCAR and AECC. Daniela Sia is supported by the Gilead Sciences Research Scholar Program in Liver Disease. Scott L. Friedman is supported by the National Institutes of Health Research project grant (R01,DK5662) and US Department of Defense (CA150272P3). Mathias Heikenwälder was supported by an ERC Consolidator grant (HepatoMetaboPath), the SFBTR 209, 1335 and SFBTR179.Background & Aims: Cirrhosis and chronic inflammation precede development of hepatocellular carcinoma (HCC) in approximately 80% of cases. We investigated immune-related gene expression patterns in liver tissues surrounding early-stage HCCs and chemopreventive agents that might alter these patterns to prevent liver tumorigenesis. Methods: We analyzed gene expression profiles of nontumor liver tissues from 392 patients with early-stage HCC (training set, N = 167 and validation set, N = 225) and liver tissue from patients with cirrhosis without HCC (N = 216, controls) to identify changes in expression of genes that regulate the immune response that could contribute to hepatocarcinogenesis. We defined 172 genes as markers for this deregulated immune response, which we called the immune-mediated cancer field (ICF). We analyzed the expression data of liver tissues from 216 patients with cirrhosis without HCC and investigated the association between this gene expression signature and development of HCC and outcomes of patients (median follow-up, 10 years). Human liver tissues were also analyzed by histology. C57BL/6J mice were given a single injection of diethylnitrosamine (DEN) followed by weekly doses of carbon tetrachloride to induce liver fibrosis and tumorigenesis. Mice were then orally given the multiple tyrosine inhibitor nintedanib or vehicle (controls); liver tissues were collected and histology, transcriptome, and protein analyses were performed. We also analyzed transcriptomes of liver tissues collected from mice on a choline-deficient high-fat diet, which developed chronic liver inflammation and tumors, orally given aspirin and clopidogrel or the anti-inflammatory agent sulindac vs mice on a chow (control) diet. Results: We found the ICF gene expression pattern in 50% of liver tissues from patients with cirrhosis without HCC and in 60% of nontumor liver tissues from patients with early-stage HCC. The liver tissues with the ICF gene expression pattern had 3 different features: increased numbers of effector T cells; increased expression of genes that suppress the immune response and activation of transforming growth factor β signaling; or expression of genes that promote inflammation and activation of interferon gamma signaling. Patients with cirrhosis and liver tissues with the immunosuppressive profile (10% of cases) had a higher risk of HCC (hazard ratio, 2.41; 95% confidence interval, 1.21-4.80). Mice with chemically induced fibrosis or diet-induced steatohepatitis given nintedanib or aspirin and clopidogrel down-regulated the ICF gene expression pattern in liver and developed fewer and smaller tumors than mice given vehicle. Conclusions: We identified an immune-related gene expression pattern in liver tissues of patients with early-stage HCC, called the ICF, that is associated with risk of HCC development in patients with cirrhosis. Administration of nintedanib or aspirin and clopidogrel to mice with chronic liver inflammation caused loss of this gene expression pattern and development of fewer and smaller liver tumors. Agents that alter immune regulatory gene expression patterns associated with carcinogenesis might be tested as chemopreventive agents in patients with cirrhosis

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. METHODOLOGY: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. PRINCIPAL FINDINGS: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. CONCLUSIONS: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively

Irradiation leads to apoptosis of Kupffer cells by a Hsp27-dependant pathway followed by release of TNF-α

In a previous publication, we were able to show that irradiation of Kupffer cells, the liver resident macrophages, leads to an increased TNF-α concentration in the culture medium. The pathomechanisms underlying this phenomenon, however, remained to be elucidated. Here, we show that following irradiation of Kupffer cells, the apoptosis rate increased drastically within 48 h. At the same time, the total TNF-α concentration in cell lysates of Kupffer cells attached to the culture plate decreased. However, normalization of the TNF-α concentration with respect to cell number revealed that TNF-α concentration per attached cell remained constant during the observation period. Western blot analysis showed that heat shock protein 27 (Hsp27) is strongly downregulated and bax is upregulated in irradiated Kupffer cells as compared to sham-irradiated cells. Overexpression of Hsp27 in Kupffer cells was shown to prevent the effect of irradiation on bax expression, apoptosis and, at the same time, on increase of TNF-α concentration in the Kupffer cell medium. We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression. Furthermore, we suggest that apoptosis of Kupffer cells leads to an increase of TNF-α concentration in the culture medium which may be due to cell death rather than active release or synthesis

Molecular characterization of hepatocellular carcinoma in patients with nonalcoholic steatohepatitis

Background and aims: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies. Methods: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays. Results: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-β proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved. Conclusions: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature. Lay summary: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC