Multimodal decoding of human liver regeneration.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

Post-Transcriptional Regulation of Hepatic DDAH1 with TNF Blockade Leads to Improved eNOS Function and Reduced Portal Pressure In Cirrhotic Rats.

Portal hypertension (PH) is a major cause of morbidity and mortality in chronic liver disease. Infection and inflammation play a role in potentiating PH and pro-inflammatory cytokines, including TNF, are associated with severity of PH. In this study, cirrhotic bile duct ligated (BDL) rats with PH were treated with Infliximab (IFX, a monoclonal antibody against TNF) and its impact on modulation of vascular tone was assessed. BDL rats had increased TNF and NFkB compared to sham operated rats, and their reduction by IFX was associated with a reduction in portal pressure. IFX treatment also reduced hepatic oxidative stress, and biochemical markers of hepatic inflammation and injury. IFX treatment was associated with an improvement in eNOS activity and increased L-arginine/ADMA ratio and DDAH1 expression. In vitro analysis of HepG2 hepatocytes showed that DDAH1 protein expression is reduced by oxidative stress, and this is in part mediated by post-transcriptional regulation by the 3'UTR. This study supports a role for the DDAH1/ADMA axis on the effect of inflammation and oxidative stress in PH and provides insight for new therapies.

Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a syndrome that occurs in cirrhosis characterized by organ failure(s) and high mortality rate. There are no biomarkers of ACLF. The LCN2 gene and its product, neutrophil gelatinase-associated lipocalin (NGAL), are upregulated in experimental models of liver injury and cultured hepatocytes as a result of injury by toxins or proinflammatory cytokines, particularly Interleukin-6. The aim of this study was to investigate whether NGAL could be a biomarker of ACLF and whether LCN2 gene may be upregulated in the liver in ACLF. METHODS: We analyzed urine and plasma NGAL levels in 716 patients hospitalized for complications of cirrhosis, 148 with ACLF. LCN2 expression was assessed in liver biopsies from 29 additional patients with decompensated cirrhosis with and without ACLF. RESULTS: Urine NGAL was markedly increased in ACLF vs. no ACLF patients (108(35-400) vs. 29(12-73)µg/g creatinine; p<0.001) and was an independent predictive factor of ACLF; the independent association persisted after adjustment for kidney function or exclusion of variables present in ACLF definition. Urine NGAL was also an independent predictive factor of 28day transplant-free mortality together with MELD score and leukocyte count (AUROC 0.88(0.83-0.92)). Urine NGAL improved significantly the accuracy of MELD in predicting prognosis. The LCN2 gene was markedly upregulated in the liver of patients with ACLF. Gene expression correlated directly with serum bilirubin and INR (r=0.79; p<0.001 and r=0.67; p<0.001), MELD (r=0.68; p<0.001) and Interleukin-6 (r=0.65; p<0.001). CONCLUSIONS: NGAL is a biomarker of ACLF and prognosis and correlates with liver failure and systemic inflammation. There is remarkable overexpression of LCN2 gene in the liver in ACLF syndrome. LAY SUMMARY: Urine NGAL is a biomarker of acute-on-chronic liver failure (ACLF). NGAL is a protein that may be expressed in several tissues in response to injury. The protein is filtered by the kidneys due to its small size and can be measured in the urine. Ariza, Graupera and colleagues found in a series of 716 patients with cirrhosis that urine NGAL was markedly increased in patients with ACLF and correlated with prognosis. Moreover, gene coding NGAL was markedly overexpressed in the liver tissue in ACLF.

Early invasive fungal infections and colonization in patients with cirrhosis admitted to the intensive care unit.

Bacterial infections in cirrhosis are common and associated with increased mortality, but little is known about fungal infections. The aim of this study, a sub-analysis of the Fungal Infection Risk Evaluation study, was to assess the incidence and implications of early invasive fungal disease (IFD) in patients with cirrhosis admitted to intensive care units (ICU). Clinical and laboratory parameters collected in the first 3 days of ICU stay for 782 patients with cirrhosis and/or portal hypertension were analysed and compared with those of 273 patients with very severe cardiovascular disease (CVD). The CVD patients had more co-morbidities and higher APACHE II scores. The overall incidence of IFD was similar in the two groups, but the incidence of IFD in ICU was higher in liver patients (1% versus 0.4%; p 0.025) as was fungal colonization (23.8% versus 13.9%; p 0.001). The ICU and in-hospital mortality, and length of stay were similar in the two groups. A higher proportion of liver patients received antifungal therapy (19.2% versus 7%; p <0.0005). There was no difference in mortality between colonized patients who received antifungal therapy and colonized patients who did not. The incidence of IFD in patients with cirrhosis in ICU is higher compared with another high-risk group, although it is still very low. This risk might be higher in patients with advanced liver disease admitted with acute-on-chronic liver failure, and this should be investigated further. Our data do not support prophylactic use of antifungal therapy in cirrhosis.

Obesity is independently associated with infection in hospitalised patients with end-stage liver disease.

BACKGROUND: Infection is the most common cause of mortality in end-stage liver disease (ESLD). The impact of obesity on infection risk in ESLD is not established. AIM: To characterise the impact of obesity on infection risk in ESLD. METHODS: We evaluated the association between infection and obesity in patients with ESLD. Patients grouped as non-obese, obesity class I-II and obesity class III were studied using the Nationwide Inpatient Sample. Validated diagnostic code based algorithms were utilised to determine weight category and infections, including bacteraemia, skin/soft tissue infection, urinary tract infection (UTI), pneumonia/respiratory infection, Clostridium difficile infection (CDI) and spontaneous bacterial peritonitis (SBP). Risk factors for infection and mortality were assessed using multivariable logistic regression analysis. RESULTS: Of 115 465 patients identified, 100 957 (87.5%) were non-obese and 14 508 (12.5%) were obese, with 9489 (8.2%) as obesity class I-II and 5019 (4.3%) as obesity class III. 37 117 patients (32.1%) had an infection diagnosis. Infection was most prevalent among obesity class III (44.0%), followed by obesity class I-II (38.9%) and then non-obese (31.9%). In multivariable modelling, class III obesity (OR = 1.41; 95% CI 1.32-1.51; P < 0.001), and class I-II obesity (OR = 1.08; 95% CI 1.01-1.15; P = 0.026) were associated with infection. Compared to non-obese patients, obese individuals had greater prevalence of bacteraemia, UTI, and skin/soft tissue infection as compared to non-obese patients. CONCLUSIONS: Obesity is newly identified to be independently associated with infection in end-stage liver disease. The distribution of infection sites varies based on weight category.

Ornithine phenylacetate targets alterations in the expression and activity of glutamine synthase and glutaminase to reduce ammonia levels in bile duct ligated rats.

BACKGROUND & AIMS: In liver failure, ammonia homeostasis is dependent upon the function of the ammonia metabolising enzymes, glutamine synthetase (GS) and glutaminase (GA) but data about their protein expression and activity are lacking. The aims of this study were to determine the protein expression and activity of GS and GA in individual organs in a rat model of chronic liver disease and to test whether the treatment with the ammonia-lowering agent ornithine phenylacetate (OP) modulates their activities. METHODS: 49 SD rats were studied 35 days after sham-operation or bile duct ligation (BDL). The BDL group received: L-ornithine (0.6 mg/kg/day), Phenylacetate (0.6 mg/kg/day), OP (0.6 mg/kg/day) or placebo (saline) for 5 days prior to sacrifice. Arterial ammonia, amino acids and liver biochemistry were measured. Expressions of GS and GA were determined by Western-blotting and activities by end-point methods in liver, muscle, gut, kidney, lung, and frontal cortex. RESULTS: In BDL rats, hepatic GS enzyme activity was reduced by more than 80% compared to sham rats. Further, in BDL rats GA activity was reduced in liver but increased in the gut, muscle and frontal cortex compared to sham rats. OP treatment resulted in a reduction in hyperammonemia in BDL rats, associated with increased GS activity in the muscle and reduced gut GA activity. CONCLUSIONS: In a rat model of chronic liver failure, hyperammonemia is associated with inadequate compensation by liver and muscle GS activity and increased gut GA activity. OP reduces plasma ammonia by increasing GS in the muscle and reducing GA activity in the gut providing additional insights into its mechanism of its action. GS and GA may serve as important future therapeutic targets for hyperammonemia in liver failure.

Overnight glucose infusion suppresses renal ammoniagenesis and reduces hyperammonaemia induced by a simulated bleed in cirrhotic patients.

BACKGROUND: A simulated upper gastrointestinal (UGI) bleed in cirrhotic patients has been shown to induce hyperammonaemia. The kidney was the site of this exaggerated ammoniagenesis with alanine as substrate. Administration of alanine to decompensated cirrhotic patients did not change hepatic gluconeogenesis, but resulted in increased ammoniagenesis. We hypothesise that reduced hepatic glycogen stores result in hyperglucagonaemia which may drive increased renal gluconeogenesis and therefore alanine uptake and renal ammoniagenesis. AIM: To determine whether an overnight glucose infusion lowers renal ammoniagenesis by reducing hyperglucagonaemia and renal ammoniagenesis. METHODS: Patients with decompensated cirrhosis were studied in a cross-over design. An UGI bleed was simulated via intragastric administration of an amino acids mixture mimicking the haemoglobin molecule after a 12-h overnight fast (F-group) or after a 12-h treatment with 20% glucose solution (G-group). RESULTS: Before the simulated bleed the glucagon levels were 21 (15-31) pmol/L in the F-group and 15 (9-21) pmol/L in the G-group (P < 0.01). After the simulated bleed, arterial ammonia levels increased in both groups [F-group: 73-118 µmol/L (P = 0.01); G-group 64-87 µmol/L (P = 0.01)]. The enhancement of hyperammonaemia was significantly higher in the F-group (45 [19-71] µmol/L) compared with the G-group (23 [13-39] µmol/L) (P = 0.01). The difference in renal ammoniagenesis during the simulated bleed in the F-group was 399 (260-655) nmol/kg/bwt/min and was significantly higher than in the G-group 313 (1-498) nmol/kg/bwt/min (P = 0.05). CONCLUSIONS: Overnight glucose infusion results in reduced renal ammoniagenesis and attenuates ammonia levels. These observations have implications for the development of nutritional strategies in hyperammonaemic patients.

Dogs with congenital porto-systemic shunting (cPSS) and hepatic encephalopathy have higher serum concentrations of C-reactive protein than asymptomatic dogs with cPSS.

Hepatic encephalopathy (HE) is a cause of significant morbidity and mortality in patients with liver disorders and a wide range of rodent models of HE have been described to facilitate studies into the pathogenesis and treatment of HE. However, it is widely acknowledged that no individual model perfectly mimics human HE and there is a particular need for spontaneous, larger animal models. One common congenital abnormality in dogs is the portosystemic shunt (cPSS) which causes clinical signs that are similar to human HE such as ataxia, disorientation, lethargy and occasionally coma. As inflammation has recently been shown to be associated with HE in humans, we hypothesised that inflammation would similarly be associated with HE in dogs with cPSS. To examine this hypothesis we measured C-reactive protein (CRP) in 30 healthy dogs, 19 dogs with a cPSS and no HE and 27 dogs with a cPSS and overt HE. There was a significant difference in CRP concentration between healthy dogs and dogs with HE (p < 0.001) and between dogs with HE and without HE (p < 0.05). The novel finding that there is an association between inflammation and canine HE strengthens the concept that HE in dogs with cPSS shares a similar pathogenesis to humans with HE. Consequently, dogs with a cPSS may be a good spontaneous model of human HE in which to further examine the role of inflammation and development of HE.

'Out-patient' albumin dialysis for cholestatic patients with intractable pruritus.

BACKGROUND: Intractable pruritus is a major problem for some patients with cholestasis. Albumin dialysis has been shown to ameliorate pruritus, but long-term outcome data are limited. AIM: To evaluate the safety and efficacy of 'out-patient' albumin dialysis using the molecular adsorbent recirculating system (MARS) in the treatment of intractable pruritus in cholestatic patients referred for liver transplantation for symptomatic relief. METHODS: Fifteen patients who failed to respond to standard medical therapy to control pruritus were included. Three MARS (6 h) sessions were performed per admission, and were repeated, if necessary. The intensity and severity of itch was quantified using itch severity and visual analogue scales (ISS and VAS). RESULTS: Molecular adsorbent recirculating system treatment was safe and associated with immediate and complete response in 11 patients; two patients had a partial response and two patients had no response. Thirty-four treatments were performed during a follow-up period of 15.7 months (3-46) with patients requiring a mean of two admissions (1-6). The mean VAS and ISS improved significantly (both P < 0.001) with improvement in the patient's perception of their quality of life. The duration of acceptable relief in responders was 3.3 months (range 2-5). No serious adverse events were recorded, but the platelet count and haemoglobin were reduced significantly. CONCLUSION: Molecular adsorbent recirculating system therapy delivered in an 'out-patient' setting is safe and effective with a high degree of patient acceptability. Albumin dialysis can be considered a viable therapeutic option for patients with severe intractable pruritus, in whom, the only other effective treatment option is liver transplantation.

Management of hepatic encephalopathy.

Hepatic encephalopathy (HE), the neuropsychiatric presentation of liver disease, is associated with high morbidity and mortality. Reduction of plasma ammonia remains the central therapeutic strategy, but there is a need for newer novel therapies. We discuss current evidence supporting the use of interventions for both the general management of chronic HE and that necessary for more acute and advanced disease.

Acute endotoxemia following transjugular intrahepatic stent-shunt insertion is associated with systemic and cerebral vasodilatation with increased whole body nitric oxide production in critically ill cirrhotic patients.

BACKGROUND & AIMS: Transjugular intrahepatic stent-shunt (TIPSS) insertion, in patients with uncontrolled gastro-intestinal bleeding, often results in worsening of the systemic hemodynamics which can be associated with intracranial hypertension but the underlying mechanisms are unclear. This study explored the hypothesis that TIPSS insertion results in acute endotoxemia which is associated with increased nitric oxide production resulting in systemic and cerebral vasodilatation. METHODS: Twelve patients with cirrhosis who were undergoing TIPSS for uncontrolled variceal bleeding were studied prior to and 1-h after TIPSS insertion. Changes in cardiac output (CO) and cerebral blood flow (CBF) were measured. NO production was measured using stable isotopes using l-[guanidino-(15)N(2)] arginine and l-[ureido-(13)C;5,5-(2)H(2)] citrulline infusion. The effect of pre- and post-TIPSS plasma on nitric oxide synthase (NOS) activity on human endothelial cell-line (HUVEC) was measured. RESULTS: TIPSS insertion resulted in a significant increase in CO and CBF. Endotoxin and induced neutrophil oxidative burst increased significantly without any significant changes in cytokines. Whole body NO production increased significantly and this was associated with increased iNOS activity in the HUVEC lines. The change in NO production correlated with the changes in CO and CBF. Brain flux of ammonia increased without significant changes in arterial ammonia. CONCLUSIONS: In conclusion, the insertion of TIPSS results in acute endotoxemia which is associated with increased nitric oxide production possibly through an iNOS dependent mechanism which may have important pathophysiological and therapeutic relevance to understanding the basis of circulatory failure in the critically ill cirrhotic patient.

Review article: hyperammonaemic and catabolic consequences of upper gastrointestinal bleeding in cirrhosis.

BACKGROUND: Upper gastrointestinal (UGI) bleeding in patients with cirrhosis of the liver induces hyperammonaemia and leads to a catabolic cascade that precipitates life-threatening complications. The haemoglobin molecule is unique because it lacks the essential amino acid isoleucine and contains high amounts of leucine and valine. UGI bleed therefore presents the gut with protein of very low biologic value, which may be the stimulus to induce net catabolism. AIM: To describe the hyperammonaemic and catabolic consequences of UGI bleeding in cirrhosis. METHODS: A semi-structured literature search was performed using PubMed and article references. RESULTS: It has recently been proven that ('simulation of ') a UGI bleed in patients with cirrhosis leads to impaired protein synthesis that can be restored by intravenous infusion of isoleucine. This may have therapeutic implications for the function of rapidly dividing cells and short half-life proteins such as clotting factors. Renal and small bowel ammoniagenesis were shown to be the most prominent causes for the hyperammonaemia that resulted from a UGI bleed. This provides an explanation for the therapeutic failure of the current clinical therapies that are aimed at large bowel-derived ammonia production. Isoleucine infusion did not diminish renal ammoniagenesis. CONCLUSIONS: New pharmacological therapies to diminish postbleeding hyperammonaemia should target the altered inter-organ ammonia metabolism and promote ammonia excretion and/or increase the excretion of precursors of ammoniagenesis, e.g. l-ornithine-phenylacetate.

Role of artificial liver support in hepatic encephalopathy.

Hepatic encephalopathy (HE) refers to the reversible neuropsychiatric disorders observed in acute liver failure and as a complication of cirrhosis and/or portal hypertension. This review aims to describe the pathophysiology of HE, the rationale for the use of artificial liver support in the treatment of HE, the different concepts of artificial liver support and the results obtained. Ammonia has been considered central to its pathogenesis but recently an important role for its interaction with inflammatory responses and auto-regulation of cerebral hemodynamics has been suggested. Artificial liver support might be able to decrease ammonia and modulate inflammatory mediators and cerebral hemodynamics. Bioartificial liver support systems use hepatocytes in an extracorporeal device connected to the patient's circulation. Artificial liver support is intended to remove protein-bound toxins and water-soluble toxins without providing synthetic function. Both systems improve clinical and biochemical parameters and can be applied safely to patients. Clinical studies have shown that artificial liver support, especially albumin dialysis, is able to improve HE in acute and acute-on-chronic liver failure. Further studies are required to better understand the mechanism, however, artificial liver support can be added to the therapeutic bundle in treating HE.

Role of Toll-like receptors 2, 4, and 9 in mediating neutrophil dysfunction in alcoholic hepatitis.

Neutrophil dysfunction in alcoholic hepatitis is associated with endotoxemia and an increased incidence of infection, but the mechanism is unclear. We aimed to investigate the role of Toll-like-receptors (TLR)2, 4, and 9 in mediating neutrophil dysfunction in alcoholic hepatitis. Neutrophils from healthy volunteers were incubated with alcoholic hepatitis patients' plasma (n = 12) with and without TLR2, 4, or 9 antagonists and with and without human albumin. TLR2, 4, and 9 expression, neutrophil oxidative burst, phagocytosis, and CXCR1+2 expression were measured by FACS analysis. Patients' plasma increased oxidative burst, decreased CXCR1+2 expression, and decreased phagocytosis of normal neutrophils in association with increased expression of TLR2, 4, and 9 and depletion of ATP. Inhibition of TLR2, 4, and 9 prevented the increase in oxidative burst and the decrease in CXCR1 and CXCR2 expression but did not prevent phagocytic dysfunction. Incubation with albumin completely prevented the patient plasma induced neutrophil dysfunction. Increased expression of TLR2, 4, and 9 is associated with neutrophil dysfunction, endotoxemia, and energy depletion. TLR2, 4, and 9 inhibition does not improve phagocytosis, indicating that TLR overexpression may be the result and not the cause of neutrophil activation. Albumin, an endotoxin scavenger, prevents the deleterious effect of patients' plasma on neutrophil phagocytosis, resting burst, and TLR expression.

The treatment of hepatic encephalopathy.

Current recommendations for the treatment of hepatic encephalopathy are based, to a large extent, on open or uncontrolled trials, undertaken in very small numbers of patients. In consequence, there is ongoing discussion as to whether the classical approach to the treatment of this condition, which aims at reducing ammonia production and absorption using either non-absorbable disaccharides and/or antibiotics, should be revisited, modified or even abandoned. Pros and cons of present therapeutic strategies and possible future developments were discussed at the fourth International Hannover Conference on Hepatic Encephalopathy held in Dresden in June 2006. The content of this discussion is summarized.

L-Ornithine phenylacetate (OP): a novel treatment for hyperammonemia and hepatic encephalopathy.

Hepatic encephalopathy (HE) is a common neuropsychiatric complication of liver disease affecting about 20-30% patients with cirrhosis. HE may only affect quality of life (e.g. impairments in attention; coordination; driving ability), but in some patients this progresses to coma and death; defining mortality in those with acute liver failure. HE is thought to occur through accumulation of ammonia as a by-product of protein metabolism. In liver failure ammonia accumulates to toxic levels, resulting in ammonia-associated brain swelling. Presently, there is no proven therapy for HE though recent studies suggest that during liver failure, ammonia removal by skeletal muscle (by conversion to glutamine) can be manipulated; also that ammonia and amino acid metabolism should be viewed in terms of their interorgan relationship. This led us to develop a novel concept for ammonia removal. Preliminary studies provide the proof of concept that the combination of L-ornithine (amino acid) with phenylactetate, as L-ornithine phenylacetate (OP), reduces toxic levels of ammonia by (1) L-ornithine acting as a substrate for glutamine synthesis from ammonia in skeletal muscle and (2) phenylacetate excreting the ornithine-related glutamine as phenylacetylglutamine in the kidneys. As both L-ornithine and phenylacetate are already available for human use, data showing its usefulness in ammonia lowering could translate quickly into providing the much needed therapy for HE patients.