Is there a role for neuregulin 4 in human nonalcoholic fatty liver disease?

BACKGROUND: Neuregulin 4 (Nrg4), a novel adipokine enriched in brown adipose tissue has been observed to negatively regulate de novo hepatic lipogenesis and limit nonalcoholic fatty liver disease (NAFLD) progression to nonalcoholic steatohepatitis (NASH) in rodents. However, the role of Nrg4 in human NAFLD remains unclear to date. We analysed Nrg4 plasma levels and its association with liver disease severity together with the transcriptional profile of the Nrg4 pathway in liver and visceral adipose tissue (VAT) of NAFLD patients. METHODS: Plasma Nrg4 levels were measured in 65 NAFLD patients and 43 healthy controls (HC). Hepatic steatosis and fibrosis were diagnosed and quantified with chemical shift MRI and transient elastography respectively. Furthermore, blood lipid levels, HOMA-IR and systemic pro-inflammatory cytokines (TNF-α, IL-6 and IFN-γ) were analysed. Microarray analyses to assess differences in the Nrg4 and its receptor family ErbB pathway in liver and VAT from an independent patient group with biopsy proven NAFL (simple steatosis) (n = 4), NASH (n = 5) and normal liver (n = 6) were performed. RESULTS: Plasma Nrg4 levels were not significantly different between NAFLD patients and HC (p = 0.622). Furthermore, plasma Nrg4 levels did not correlate with the hepatic fat fraction (r = -0.028, p = 0.829) and were not significantly different between NAFLD patients with or without hepatic fibrosis (p = 0.087). Finally, the expression profile of 82 genes related to the Nrg4-ErbB pathway in liver and VAT was not significantly different between NAFL, NASH or obese controls. CONCLUSION: Our study does not support a role for Nrg4 in the pathophysiology of human NAFLD.

Stimulation of reactive oxygen species and collagen synthesis by angiotensin II in cardiac fibroblasts.

Superoxide anion generated by NAD(P)H-oxidase has an important role in the pathogenesis of cardiovascular diseases and scavenging superoxide anion can be considered as a reasonable therapeutic strategy. In hypertensive heart diseases there is a mutual reinforcement of reactive oxygen species (ROS) and angiotensin II (ANG II). ANG II increases the NAD(P)H-dependent superoxide anion production and the intracellular generation of ROS in cardiac fibroblasts and apocynin, a membrane NAD(P)H oxidase inhibitor, abrogates this rise. ANG II also stimulates the collagen production, the collagen I and III content and mRNA expression in cardiac fibroblasts and apocynin abolishes this induction. In this review we demonstrate that scavenging superoxide anion by tempol or EUK-8 or administration of PEG-superoxide dismutase (SOD) inhibits collagen production in cardiac fibroblasts. On the contrary increasing superoxide anion formation by inhibition of SOD stimulates collagen production. A vital role of SOD and the generated ROS can be suggested in the regulation and organization of collagen in cardiac fibroblasts. Specific pharmacological intervention with SOD mimetics can probably be an alternative approach for reducing myocardial fibrosis.

Tumor initiation and progression in hepatocellular carcinoma: risk factors, classification, and therapeutic targets.

Hepatocellular carcinoma (HCC) is a major health problem worldwide responsible for 500 000 deaths annually. A number of risk factors are associated with either the induction of the disease or its progression; these include infection with hepatitis B or C virus, alcohol consumption, non-alcoholic steatohepatitis and certain congenital disorders. In around 80% of the cases, HCC is associated with cirrhosis or advanced fibrosis and with inflammation and oxidative stress. In this review we focus firstly on the different risk factors for HCC and summarize the mechanisms by which each is considered to contribute to HCC. In the second part we look at the molecular processes involved in cancer progression. HCC development is recognized as a multistep process that normally develops over many years. Over this period several mutations accumulate in the cell and that stimulate malign transformation, growth, and metastatic behavior. Over the recent years it has become evident that not only the tumor cell itself but also the tumor microenviroment plays a major role in the development of a tumor. There is a direct link between the role of inflammation and cirrhosis with this microenviroment. Both in vitro and in vivo it has been shown that tumor formation and metastatic properties are linked to epithelial-mesenchymal transition (EMT), a process by which facillitates the tumor cell's attempts to migrate to a more favourable microenviroment. Several groups have analyzed the gene expression in HCC and its surrounding tissue by microarray and this has resulted in the molecular classification into a distinct number of classes. Here we also found a role for hypoxia induced gene expression leading to a clinically more aggressive gene expression in HCC. Molecular analysis also helped to identify important cellular pathways and possible therapeutic targets. The first molecule that in this way has shown clinical application for liver cancer is the multikinase inhibitor sorafenib, others are currently in different stages of clinical studies like the mTOR inhibitor everolimus.

Downregulation of manganese superoxide dismutase by angiotensin II in cardiac fibroblasts of rats: Association with oxidative stress in myocardium.

BACKGROUND: The aim of this study was to determine whether angiotensin II (ANG II) affects the protein and mRNA expressions of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) in cardiac fibroblasts of rats through inducing the phosphorylation of the proteins Akt and FOXO3a, thereby contributing to the oxidative stress in the myocardium. METHODS: Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in serum-free Dulbecco's modified Eagle's medium for 24 h. The cells were then preincubated with/without the tested inhibitors for 1 h and then further incubated with/without ANG II (1 µmol/l) for 24 h. RESULTS: ANG II increased the production of superoxide ions in the cardiac fibroblasts, and decreased the activity levels of both Mn-SOD and CuZn-SOD, but not the activity levels of catalase and glutathione peroxidase. ANG II also decreased the mRNA and protein expressions of Mn-SOD, but not those of CuZn-SOD, catalase, and glutathione peroxidase. The likely mechanism through which ANG II produces the effect of reducing Mn-SOD activity is by reducing the extent of binding of FOXO3a to the Mn-SOD promoter. In control fibroblasts, inhibition of FOXO3a transcription with small-interfering RNA (siRNA) led to a reduction in the binding of FOXO3a to the Mn-SOD promoter, and a concomitant reduction in Mn-SOD gene expression. Our data suggest that when Akt is phosphorylated by ANG II, P-Akt is translocated from the cytoplasm to the nucleus; subsequently, nuclear phosphorylation of FOXO3a by P-Akt leads to relocalization of FOXO3a from the nucleus to the cytosol, resulting in a decrease in its transcriptional activity, and consequently in Mn-SOD expression. The likelihood of such a mechanism of action is further strengthened by the fact that inhibition of phosphoinositide 3-kinase with wortmannin or LY 294002, and Akt inhibition, were shown to lead to a decrease in P-AKT and to a consequent increase in Mn-SOD mRNA expression. CONCLUSIONS: Our data indicate that ANG II inactivates FOXO3a by activating Akt, leading to a reduction in the expression of the antioxidant Mn-SOD, and thereby potentially contributing to oxidative stress in the myocardium.

Increased serum phospholipase A2 activity after non-heart-beating donor liver transplantation and association with ischemia-reperfusion injury.

BACKGROUND: Secretory phospholipase A(2) (sPLA(2)) degrades cell membrane phospholipids and plays an important role in the synthesis of pro-inflammatory lipid mediators (arachidonic acid and cytokines) during inflammatory events such as ischemia-reperfusion injury after liver transplantation (LTx). A role for sPLA(2) in LTx from non-heart-beating donors (NHBD) has never been demonstrated. Furthermore data on the natural sPLA(2) inhibition capacity are scarce. MATERIALS AND METHODS: Using our previously validated pig model of NHBD-LTx, we evaluated changes in sPLA(2) enzyme activity in serum early after reperfusion of livers exposed to warm ischemia. Porcine livers were exposed to incremental periods of warm ischemia, procured, and transplanted after 4 h of cold storage. In serum samples, collected prior to and after reperfusion, sPLA(2) enzyme activity, tumor necrosis factor-alpha, and interleukin-6 levels were determined. RESULTS: After reperfusion, sPLA(2) activity increased and peaked significantly at 60 min in recipients with primary nonfunction (PNF). Tumor necrosis factor-alpha and interleukin-6 peaked later (at 180 min) in these PNF recipients. We observed a strong natural inhibition of sPLA(2) activity in serum at baseline; this inhibition was substantial reduced 1 h after reperfusion in both PNF and non-PNF recipients. In the non-PNF recipients, however, this natural PLA(2) inhibition was restored within 24 h after reperfusion. CONCLUSIONS: This study suggests that an increased sPLA(2) activity and a reduced inhibition may play an important role in the pathogenesis of ischemia-reperfusion injury of NHBD liver grafts.

TGF-beta1-induced cardiac myofibroblasts are nonproliferating functional cells carrying DNA damages.

TGF-beta1 induces differentiation and total inhibition of cardiac MyoFb cell division and DNA synthesis. These effects of TGF-beta1 are irreversible. Inhibition of MyoFb proliferation is accompanied with the expression of Smad1, Mad1, p15Ink4B and total inhibition of telomerase activity. Surprisingly, TGF-beta1-activated MyoFbs are growth-arrested not only at G1-phase but also at S-phase of the cell cycle. Staining with TUNEL indicates that these cells carry DNA damages. However, the absolute majority of MyoFbs are non-apoptotic cells as established with two apoptosis-specific methods, flow cytometry and caspase-dependent cleavage of cytokeratin 18. Expression in MyoFbs of proliferative cell nuclear antigen even in the absence of serum confirms that these MyoFbs perform repair of DNA damages. These results suggest that TGF-beta1-activated MyoFbs can be growth-arrested by two checkpoints, the G1/S checkpoint, which prevents cells from entering S-phase and the intra-S checkpoint, which is activated by encountering DNA damage during the S phase or by unrepaired damage that escapes the G1/S checkpoint. Despite carrying of the DNA damages TGF-beta1-activated MyoFbs are highly functional cells producing lysyl oxidase and contracting the collagen matrix.

HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development.

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H(2)O(2), the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H(2)O(2) to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O(2)(-)) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H(2)O(2) directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.

Viral interaction and clinical implications of coinfection of hepatitis C virus with other hepatitis viruses.

Coinfection with other hepatitis viruses modifies the viral profile in serum and leads to more liver damage and more rapid progression during the course of hepatitis C virus infection. The viral interference is not only carried out by virus-virus or by virus-cell interactions but also by an enhanced immune response. A superinfecting viral infection does not crossactivate protective immune responses to the pre-existing virus albeit the latter can become undetectable. The induced cytokine stimulation might enhance the hepatic inflammation. Moreover, hepatitis B virus coinfection increases the risk of development of hepatocellular carcinoma in hepatitis C virus patients through common necro-inflammatory pathways or by direct oncogenic activity of hepatitis B virus. Viral interaction also complicates the management of the coinfection because hepatitis C virus impairs the humoral response to hepatitis A virus and hepatitis B virus vaccines, and because pharmacological suppression of hepatitis C virus endangers dually infected patients with reactivation of coinfected hepatitis B virus. Optimized strategies and follow-up are thus necessary in the treatment of infection with multiple viruses. It seems thus necessary to look for markers of hepatitis B virus and/or hepatitis D virus infection in chronic hepatitis patients positive for hepatitis C virus antibodies but negative for hepatitis C virus RNA, and equally well to search for hepatitis C virus RNA in HBsAg-negative/anti-HBc-positive patients with a low level of serum hepatitis B virus DNA.

Hepatitis B virus replication causes oxidative stress in HepAD38 liver cells.

UNLABELLED: We used human hepatoma HepAD38 cells, in which HBV production is under the control of a tetracycline-regulated promotor, to investigate changes induced in the host cell by HBV replication that could contribute to malignant transformation. Parameters of oxidative stress (malondialdehyde, glutathione) and cell proliferation were determined at different times after induction (0-96 h). In HBV-producing cells, the redox status peaked at 72 h. cDNA micro array analysis at 72 h post induction revealed 3 groups of genes that were up-regulated by HBV: (i) heat shock proteins, (ii) oxidative and metabolic stress and (iii) growth and apoptosis related genes. Continuous HBV production did not accelerate karyotypic changes in cells cultured for 4 months (18 passages). IN CONCLUSION: HBV replication modulates host gene expression and induces oxidative stress. In this HepAD38 model early events (0-4 days) in the host cell after induction of HBV replication can be studied under strictly defined conditions.

Halofuginone can worsen liver fibrosis in bile duct obstructed rats.

BACKGROUND/AIMS: Halofuginone (HF) is an antifibrotic agent in rat models of liver fibrosis caused by repetitive intoxications. A beneficial effect of HF on a biliary type of liver fibrosis has not been proven yet. METHODS: Bile duct-obstructed rats were given HF from the moment of obstruction onwards and compared with no treatment. After 3 weeks, respectively, 6 weeks, aminopyrine breath test (ABT) and haemodynamic measurements including of portal pressure were carried out. Liver pieces were taken for Sirius red quantitative scoring, as well as for semiquantitative determinations of collagen type I and III RNA levels. RESULTS: ABT was significantly worse in HF-treated rats as compared with no treatment (P=0.02). Haemodynamic data and collagen type I and III determinations were not significantly different between groups. Biliary fibrosis scores were significantly higher in HF-treated rats as compared with no treatment (P=0.03). More Sirius red staining was associated with more proliferation of bile ductules. CONCLUSIONS: HF may worsen biliary fibrosis. This contrasts sharply with antifibrotic effects in other models of liver fibrosis. Distinctive cellular mechanisms in biliary fibrosis may explain this discrepancy. One should be cautious for chronic application of HF in man with cholestasis.

TT virus infection in acute and chronic liver diseases and in patients regularly receiving blood products in Belgium.

BACKGROUND: TT viruses are single-stranded DNA viruses, suggested to be involved in non A-E hepatitis. We studied the prevalence of TTV infection in acute or chronic hepatitis in Belgium in comparison with that in blood donors and in patients regularly receiving blood products. METHODS: TTV-DNA was detected by PCR using the primer set of Takahashi et al (1998) or a nested-PCR specific for genotype-2, because it had been reported that this subtype might be more pathogenic (Tagger et al. 1999). RESULTS: TTV-DNA was present in 49% of 128 patients with chronic hepatitis C, in 54% of 54 with chronic hepatitis B and in 54% of 24 with acute liver failure. This prevalence is similar to the 47% in 127 patients with clotting disorders, or the 64% in 103 undergoing chronic haemodialysis, but lower than the 29.7% found in 340 healthy blood donors. Significant differences in clinical or biochemical characteristics between TTV- positive or TTV-negative patients could not be substantiated. The genotype-2 subgroup comprised 3.9%, but they also did not differ from non genotype-2 patients. CONCLUSIONS: The prevalence of TTV infection was higher in patients than in healthy blood donors. Its clinical significance remains questionable since clinical and biochemical characteristics were not different between TTV positive and TTV negative patients. The higher prevalence of TTV in patients might be related to parenteral transmission, but the relatively high prevalence in healthy blood donors points to an additional presumably faeco-oral infection. The presence of TTV in animals suggests that infection might also originate from food. Long term follow-up will have to define whether co-infection with TTV eventually alters the natural history of chronic hepatitis.

Expression of hepatitis C virus core protein impairs DNA repair in human hepatoma cells.

Several studies have documented the important association between hepatitis C virus (HCV) infection and hepatocellular carcinoma. The mechanisms involved are still unknown and could involve viral proteins. We investigated the effect of HCV-core protein on DNA repair after UV-induced DNA damage. Therefore, we developed and characterized stably transfected HepG2 cell lines that express HCV-core protein as demonstrated by immunohistochemistry. These cells were significantly less capable to repair the DNA damage than control cells. This suppression of DNA repair by HCV-core protein renders the cells more sensitive to acquire mutations that in combination with enhanced in vivo cell turnover in the infected liver might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol, etc.). Interestingly, expression of the full-length HCV core did increase the cell doubling time in one of the cell lines we had developed that could not be attributed to an increase in apoptosis or change in telomerase activity in these cells.

Primary human hepatocytes are protected against prolonged and repeated exposure to ethanol by silibinin-dihemisuccinate.

AIMS AND METHODS: We investigated the effect of silibinin-C-2',3'-dihydrogensuccinate (SDH) on primary human hepatocytes when exposed to ethanol for 14 days. At regular intervals, the medium was refreshed and liver enzymes and secreted protein in the medium were determined. RESULTS: The ethanol-induced release of lactate dehydrogenase (at 34 mM ethanol) was completely blocked by 20 microM SDH. SDH itself stimulated fibrinogen release and had no toxic effect. CONCLUSIONS: We can conclude that SDH has a beneficial effect on human hepatocytes when exposed to ethanol in vitro.

Identification of HepG2 variant cell lines by short tandem repeat (STR) analysis.

In the past years, in our laboratory, several cell lines have been generated starting from a human liver (H7). Some of them have been used successfully in studies of the infection with and propagation of Hepatitis B and Hepatitis C viruses. Recently, several lines of evidence indicated that the origin of these cell lines was uncertain. Therefore, we now have determined the genetic characteristics of these cell lines in comparison to HepG2 cells received from ATCC and to HepG2 isolates grown at other laboratories. Quadruplex fluorescent short tandem repeat (STR) typing and karyotyping were performed. In addition, some biochemical characteristics of selected clones were studied. Genetically, all H7-derived cell lines were identical to HepG2 cells. However, some liver-specific functions varied between the different sub-cloned lines. The H7-derived cell lines that were generated proved to be sub-cloned lines of HepG2. The problem of cross-contamination during cloning of cell lines appears to be not uncommon. We found that two out of six HepG2 isolates obtained from other laboratories were not derived from the same individual as the original HepG2 cells. Therefore, STR typing should be applied as a rapid and sensitive technique to determine and monitor the origin of cell lines and to safeguard against contamination.

Low NO bioavailability in CCl4 cirrhotic rat livers might result from low NO synthesis combined with decreased superoxide dismutase activity allowing superoxide-mediated NO breakdown: A comparison of two portal hypertensive rat models with healthy controls.

BACKGROUND: In cirrhotic livers, the balance of vasoactive substances is in favour of vasoconstrictors with relatively insufficient nitric oxide. Endothelial dysfunction has been documented in cirrhotic rat livers leading to a lower activity of endothelial nitric oxide synthase but this might not be sufficient to explain the low nitric oxide presence. We compared the amount of all nitric oxide synthase isoforms and other factors that influence nitric oxide bioavailability in livers of two portal hypertensive rat models: prehepatic portal hypertension and carbon tetrachloride induced cirrhosis, in comparison with healthy controls. RESULTS: Endothelial nitric oxide synthase was the solely detected isoform by Western blotting in all livers. In cirrhotic livers, the amount of endothelial nitric oxide synthase protein was lower than in healthy controls, although an overlap existed. Levels of caveolin-1 messenger RNA were within the normal range but endothelin-1 messenger RNA levels were significantly higher in cirrhotic livers (p < 0.05). A markedly lower superoxide dismutase activity was observed in cirrhotic livers as compared to healthy controls (p < 0.05). CONCLUSIONS: In contrast to prehepatic portal hypertension, cirrhotic livers had decreased endothelial nitric oxide synthase protein and enhanced endothelin-1 messenger RNA amount. We hypothesise that a vasodilator/vasoconstrictor imbalance may be further aggravated by the reduced activity of superoxide dismutase. Decreased activity allows enhanced superoxide action, which may lead to breakdown of nitric oxide in liver sinusoids.