Aloysious Aravinthan

Hepatocyte senescence predicts progression in non-alcohol-related fatty liver disease

BACKGROUND & AIMS Models of non-alcohol-related fatty liver disease (NAFLD) reveal features of accelerated ageing, such as impaired regeneration, and an increased risk of hepatocellular carcinoma. The relation between accelerated ageing, disease progression and clinical outcome has not been previously investigated and is the subject of the current study. METHODS Liver sections from 70 patients with NAFLD (105 biopsies) and 60 controls were studied for telomere length, nuclear area, DNA damage and cell cycle phase markers, using quantitative fluorescent in situ hybridization and immunohistochemistry. RESULTS Hepatocyte telomeres were shorter in NAFLD than controls (p <0.0001). Hepatocytes in NAFLD demonstrated lack of cell cycle progression beyond G1/S phase and high-level expression of p21, the universal cell cycle inhibitor (p=0.001). γ-H(2)AX expression increased with steatosis (p=0.01), indicating DNA damage, and was associated with shorter hepatocyte telomeres (p <0.0001). Hepatocyte p21 expression correlated with fibrosis stage and diabetes mellitus, independently (p <0.001 and p=0.002, respectively). Further analysis revealed that an adverse liver-related outcome was strongly associated with higher hepatocyte p21 expression and greater hepatocyte nuclear area (p=0.02 and p=0.006), but not with telomere length. In paired biopsies, changes in hepatocyte p21 expression and nuclear area mirrored changes in fibrosis stage (p=0.01 and p=0.006, respectively). CONCLUSIONS These findings are consistent with hepatocyte senescence and permanent cell cycle arrest in NAFLD. Hepatocyte senescence correlated closely with fibrosis stage, diabetes mellitus, and clinical outcome. Hepatocyte p21 expression could be used as a prognostic marker and for stratification in clinical studies.

The Role Of Chronic Norovirus Infection In The Enteropathy Associated With Common Variable Immunodeficiency

OBJECTIVES:A severe enteropathy of unknown etiology can be associated with common variable immunodeficiency (CVID).METHODS:Stool and archived small intestinal mucosal biopsies from patients with CVID enteropathy were analyzed by PCR for the presence of Norovirus RNA. The PCR products were sequenced to determine the relationship of viral isolates. Stool samples from 10 patients with CVID but no enteropathy served as controls.RESULTS:All eight patients in our CVID cohort with enteropathy showed persistent fecal excretion of Norovirus. Analysis of archived duodenal biopsies revealed a strong association between the presence of Norovirus and villous atrophy over a period of up to 8 years. Analysis of the viral isolates from each patient revealed distinct strains of genogroup II.4. Sequence analysis from consecutive biopsy specimens of one patient demonstrated persistence of the same viral strain over a 6-year period. CVID patients without enteropathy showed no evidence of Norovirus carriage. Viral clearance occurred spontaneously in one patient and followed oral Ribavirin therapy in two further patients, and resulted in complete symptomatic and histological recovery. However, Ribavirin treatment in two further patients was unsuccessful.CONCLUSIONS:Norovirus is an important pathogen for patients with CVID and a cause of CVID enteropathy, as viral clearance, symptom resolution, and histological recovery coincide. Ribavirin requires further evaluation as a potential therapy.

Hepatocyte Expression of the Senescence Marker p21 Is Linked to Fibrosis and an Adverse Liver-Related Outcome in Alcohol-Related Liver Disease

Background and Aim Alcohol-related liver disease (ALD) remains a leading cause of liver-related morbidity and mortality. Age, fibrosis stage, MELD score and continued alcohol consumption predict outcome in everyday clinical practice. In previous studies increased hepatocyte nuclear area and hepatocyte expression of p21, both markers of senescence, were associated with increased fibrosis stage and a poor outcome in non-alcohol-related fatty liver disease, while increased hepatocyte nuclear area was related to liver dysfunction in ALD cirrhosis. This study, therefore, investigated the pattern of hepatocyte cell cycle phase distribution and hepatocyte p21 expression in relation to outcome in ALD. Methods Liver sections from two cohorts were studied. The first comprised 42 patients across the full spectrum of ALD. The second cohort comprised 77 patients with ALD cirrhosis. Immunohistochemistry assessed hepatocyte expression of cell cycle phase markers and p21. Regenerating liver (n=12) and “normal” liver sections (n=5) served as positive and negative controls, respectively. Results In the first cohort there was little cell cycle progression beyond G1/S phase and increased hepatocyte p21 expression (p<0.0001), which correlated independently with fibrosis stage (p=0.005) and an adverse liver-related outcome (p=0.03). In the second cohort, both hepatocyte p21 expression (p<0.001) and MELD score (p=0.006) were associated independently with an adverse liver-related outcome; this association was stronger with hepatocyte p21 expression (AUROC 0.74; p=0.0002) than with MELD score (AUROC 0.59; p=0.13). Further, hepatocyte p21 expression co-localised with increased hepatic stellate cell activation. Conclusions The findings are consistent with impaired cell cycle progression beyond the G1/S phase in ALD. The striking independent associations between increased hepatocyte p21 expression and both fibrosis stage and an adverse liver-related outcome in both cohorts suggests hepatocyte senescence plays an important role in ALD. Measuring hepatocyte p21 expression is simple and cheap and in this series was a useful measure of long-term prognosis in ALD.

Gene polymorphisms of cellular senescence marker p21 and disease progression in non-alcohol-related fatty liver disease

Non-alcohol-related fatty liver disease (NAFLD) encompasses a wide spectrum, ranging from steatosis alone to steatohepatitis and fibrosis. Presence of steatohepatitis and fibrosis are key hallmarks of disease progression. Previous studies have demonstrated an association between hepatocyte p21 expression and fibrosis stage in NAFLD. The aim of this study is to investigate the association between the variants of CDKN1A, which encodes p21, and disease progression in NAFLD. To this end, the relation between CDKN1A polymorphism and liver fibrosis was studied in 2 cohorts of biopsy-proven NAFLD patients from UK (n = 323) and Finland (n = 123). Genotyping was performed using DNA isolated from lymphocytes collected at the time of liver biopsy. The findings of the UK cohort were tested in the Finnish cohort. Both the UK and Finnish cohorts were significantly different from each other in basic demographics. In the UK cohort, rs762623, of the 6 SNPs across CDKN1A tested, was significantly associated with disease progression in NAFLD. This association was confirmed in the Finnish cohort. Despite the influence on fibrosis development, SNPs across CDKN1A did not affect the progression of liver fibrosis. In conclusion, CDKN1A variant rs762623 is associated with the development but not the propagation of progressive liver disease in NAFLD.

The senescent hepatocyte gene signature in chronic liver disease

Hepatocyte senescence is associated closely with fibrosis stage and an adverse outcome in chronic liver disease, but it is uncertain whether there is a causal relation with clinical manifestations of chronic liver disease, which was the subject of this study of the senescent hepatocyte gene signature. Senescence was induced in HepG2 cells using sub-lethal concentrations of H2O2. Gene expression of control and senescent HepG2 cells were studied. Comparison was made with patients with cirrhosis and three public microarray datasets. H2O2-treated HepG2 cells demonstrated characteristic cellular senescence. There was differential expression of 354 genes in senescence. Up-regulated genes in HepG2 senescence were also up regulated in patients with cirrhosis. The senescent hepatocyte gene signature distinguished liver disease from normal by unsupervised clustering in the public chronic liver disease microarray datasets, with enrichment of the senescence gene signature in all three datasets. The senescent hepatocyte gene signature included changes in cell cycle regulation, morphology, inflammation, signal transduction, metabolism and stellate cell activation, which alongside impaired synthetic function in senescence in vitro were consistent with manifestations of clinical liver disease, suggesting a close relation between hepatocyte senescence and manifestations of chronic liver disease including fibrosis and impaired synthetic function.

Selective insulin resistance in hepatocyte senescence.

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence.

Senescence in chronic liver disease: Is the future in aging?

Cellular senescence is a fundamental, complex mechanism with an important protective role present from embryogenesis to late life across all species. It limits the proliferative potential of damaged cells thus protecting against malignant change, but at the expense of substantial alterations to the microenvironment and tissue homeostasis, driving inflammation, fibrosis and paradoxically, malignant disease if the process is sustained. Cellular senescence has attracted considerable recent interest with recognition of pathways linking aging, malignancy and insulin resistance and the current focus on therapeutic interventions to extend health-span. There are major implications for hepatology in the field of fibrosis and cancer, where cellular senescence of hepatocytes, cholangiocytes, stellate cells and immune cells has been implicated in chronic liver disease progression. This review focuses on cellular senescence in chronic liver disease and explores therapeutic opportunities.

Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis.

Background Chronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase. Methods Liver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro. Results 13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to 
in vitro induction of cellular senescence, which had no effect. Conclusion Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence.

Vacuolation in hepatocyte nuclei is a marker of senescence

Hepatocyte nuclear vacuolation is considered benign and associated with non-alcohol-related fatty liver disease. Vacuolated hepatocyte nuclei were compared with non-vacuolated hepatocyte nuclei in eight patients with advanced fibrosis and a spectrum of liver disease to explore the hypothesis that such nuclei represent senescence. Age- and sex-matched liver donors served as normal tissue. In normal liver <0.01% hepatocytes showed nuclear vacuolation. In contrast, nuclear vacuolation was present in all patients with liver disease, ranging from 0.1% to 11.7% hepatocytes, irrespective of the aetiology of liver disease and independent of insulin resistance. There was a close association between nuclear vacuolation and increased nuclear area, p21 expression, γH2AX expression and the absence of Mcm-2, consistent with senescence and cell cycle arrest. Nuclear vacuolation in hepatocytes is a marker of senescence and likely to be a consequence of liver injury, unrelated to insulin resistance.

Relative adrenal insufficiency in a patient with liver disease.

Patients with established cirrhosis are at increased risk of sepsis. Bacterial infections are a frequent cause of morbidity and mortality in patients with advanced liver disease. Mortality for patients admitted to hospital with bacterial infection is approximately 30%, whereas the development of septic shock and multiorgan failure is associated with a mortality of 70-100%. Activation of the hypothalamic-pituitary-adrenal axis is an important feature of a patients response to severe sepsis and major trauma. An inadequate adrenal response with suboptimal cortisol production has been recognized in patients with septic shock. Patients with septic shock and adrenal insufficiency have reduced response to vasoconstrictor agents, higher rates of refractory shock and high mortality rates. An improvement in survival with administration of hydrocortisone in patients with septic shock and an inadequate adrenal response has been demonstrated. In a more recent study, however, there was no survival benefit in septic shock though reversal of shock was faster with hydrocortisone administration. Recently, adrenal insufficiency has been demonstrated in patients with severe liver disease such as acute liver failure, acute on chronic liver failure, recent liver transplantation and cirrhosis irrespective of the presence of sepsis. Nevertheless survival benefit with administration of hydrocortisone has only been demonstrated in patients with cirrhosis and septic shock. A case report of a patient with cirrhosis and adrenal insufficiency is presented with a review of the literature.