Harry Millward-Sadler

Hepatic changes in the failing Fontan circulation

Background: The failing Fontan circulation is associated with hepatic impairment. The nature of this liver injury is poorly defined. Objective: To establish the gross and histological liver changes of patients with Fontan circulation relative to clinical, biochemical and haemodynamic findings. Methods: Patients were retrospectively assessed for extracardiac Fontan conversion between September 2003 and June 2005, according to an established clinical protocol. Twelve patients, mean age 24.6 (range 15.8–43.4) years were identified. The mean duration since the initial Fontan procedure was 14.1 (range 6.9–26.4) years. Results: Zonal enhancement of the liver (4/12) on CT was more common in patients with lower hepatic vein pressures (p = 0.007), and in those with absent cardiac cirrhosis on histological examination (p = 0.033). Gastro-oesophageal varices (4/12) were more common in patients with higher hepatic vein pressure (21 (6.3) vs 12.2 (2.2) mm Hg, p = 0.013) and associated with more advanced cirrhosis (p = 0.037). The extent of cirrhosis (7/12) was positively correlated with the hepatic vein pressure (r = 0.83, p = 0.003). A significant positive correlation was found between the Fontan duration and the degree of hepatic fibrosis (r = 0.75, p = 0.013), as well as presence of broad scars (r = 0.71, p = 0.021). Protein-losing enteropathy (5/12) occurred more frequently in patients with longer Fontan duration (11.7 (3.2) vs 17.9 (6.1) years, p = 0.038). Conclusions: Liver injury, which can be extensive in this patient group, is related to Fontan duration and hepatic vein pressures. CT scan assists non-invasive assessment. Cardiac cirrhosis with the risk of developing gastro-oesophageal varices and regenerative liver nodules, a precursor to hepatocellular carcinoma, is common in this patient group.

Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration

Collagen‐I, which predominates in the neomatrix of fibrotic liver, regulates hepatocyte and hepatic stellate cell (HSC) phenotypes. Recovery from liver fibrosis is accompanied by hepatocyte regeneration, matrix degradation, and HSC apoptosis. Using mice bearing a mutated collagen‐I gene (r/r mice), which confers resistance to collagenase degradation, we have investigated the hypothesis that collagen‐I degradation is critical to HSC apoptosis and hepatocyte regeneration during recovery from liver fibrosis. During a 28‐day recovery period after 8 wk of CCl4 treatment, wild‐type (WT) livers had significantly (43%) decreased hydroxyproline (OHP) content. In r/r livers, however, OHP content remained elevated at peak fibrosis levels. Expressed markers of activated HSC (α‐smooth muscle actin, collagen‐I), elevated at peak fibrosis, dropped to control levels in WT livers after 28 days but remained raised in the r/r livers. Moreover, relative to WT livers, r/r livers had significantly reduced stellate cell apoptosis and hepatocyte regeneration during the recovery period. Using extracted collagen‐I from each genotype as culture substrata, relative to r/r, we show that WT collagen‐I promotes hepatocyte proliferation via stimulation of integrin αvβ3. Failure to degrade collagen‐I critically impairs HSC apoptosis and may prevent the effective restoration of hepatocyte mass in liver fibrosis.

Hepatic fibrosis and cirrhosis in the Fontan circulation: a detailed morphological study

Aims: To describe the histological features of the liver in patients with a Fontan circulation. Methods: Specimens from liver biopsies carried out as part of preoperative assessment prior to extracardiac cavopulmonary conversion of an older style Fontan were examined and scored semi-quantitatively for pertinent histological features. To support the use of the scoring, biopsy specimens were also ranked by eye for severity to allow correlation with assigned scores. Results: Liver biopsy specimens from 18 patients with a Fontan circulation were assessed. All specimens showed sinusoidal fibrosis. In 17 cases there was at least fibrous spur formation, with 14 showing bridging fibrosis and 2 showing frank cirrhosis. In 17 cases at least some of the dense or sinusoidal fibrosis was orcein positive, although a larger proportion of the dense fibrous bands were orcein positive compared with the sinusoidal component. All specimens showed marked sinusoidal dilatation, and 14 showed bile ductular proliferation; 1 showed minimal iron deposition, and 1 showed mild lobular lymphocytic inflammation. There was no cholestasis or evidence of hepatocellular damage. Similar appearances were observed in 2 patients with severe tricuspid regurgitation. Discussion: The histological features of the liver in patients with a Fontan circulation are similar to those described in cardiac sclerosis. Sinusoidal dilatation and sinusoidal fibrosis are marked in the Fontan series. The presence of a significant amount of orcein negative sinusoidal fibrosis suggests there may be a remediable component, although the dense fibrous bands are predominantly orcein positive, suggesting chronicity and permanence. No inflammation or hepatocellular damage is evident, suggesting that fibrosis may be mediated by a non-inflammatory mechanism.

Nuclear Factor-κB1 (p50) Limits the Inflammatory and Fibrogenic Responses to Chronic Injury

In this study we addressed the role of the nuclear factor (NF)-kappaB1/p50 subunit in chronic injury of the liver by determining the inflammatory and fibrotic responses of nfkappab1-null mice in an experimental model that mimics chronic liver disease. Mice received repeated hepatic injuries throughout 12 weeks by intraperitoneal injection of the hepatotoxin carbon tetrachloride. In response nfkappab1(-/-) mice developed more severe neutrophilic inflammation and fibrosis compared to nfkappab1(+/+) mice. This phenotype was associated with elevated hepatic expression of tumor necrosis factor (TNF)-alpha, which was localized to regions of the liver associated with inflammation and fibrosis. Hepatic stellate cells are important regulators of hepatic inflammatory and fibrogenic events but normally do not express TNF-alpha. Hepatic stellate cells derived from nfkappab1(-/-) mice expressed TNF-alpha promoter activity, mRNA, and protein. By contrast the expression of other NF-kappaB-responsive genes (ICAM1 and interleukin-6) was similar between nfkappab1(-/-) and nfkappab1(+/+) cells. We provide experimental evidence that the inappropriate expression of TNF-alpha by nfkappab1(-/-) cells is because of lack of a p50-dependent histone deacetylase 1 (HDAC1)-mediated repression of TNF-alpha gene transcription. Taken together these data indicate that the p50 NF-kappaB subunit plays a critical protective role in the injured liver by limiting the expression of TNF-alpha and its recruitment of inflammatory cells.

Arterialised hepatic nodules in the Fontan circulation: hepatico-cardiac interactions.

Hypervascular nodules occur commonly when there is hepatic venous outlet obstruction. Their nature and determinants in the Fontan circulation is poorly understood. We reviewed the records of 27 consecutive Fontan patients who had computerized tomography scan (CT) over a 4 year period for arterialised nodules and alterations in hepatic flow patterns during contrast enhanced CT scans and related these findings to cardiac characteristics. Mean patient age was 24 ± 5.8 years, (range 16.7-39.8) and mean Fontan duration was 16.8 ± 4.8 years (range 7.3-28.7). Twenty-two patients demonstrated a reticular pattern of enhancement, 4 a zonal pattern and only 1 demonstrated normal enhancement pattern. Seven (26%) patients had a median of 4 (range 1-22) arterialised nodules, mean size 1.8 cm (range 0.5 to 3.2 cm). All nodules were located in the liver periphery, their outer aspect lying within 2 cm of the liver margin. Patients with nodules had higher mean RA pressures (18 mmHg ± 5.6 vs. 13 mmHg ± 4, p=0.025), whereas their mixed venous saturation and aortic saturation was not significantly different (70% ± 11 vs. 67% ± 9 and 92% ± 10 vs. 94% ± 4, p>0.05). Post-mortem histology suggests focal nodular hyperplasia is the underlying pathology. ConclusionsAbnormalities of hepatic blood flow and the presence of arterialised nodules are common in the failing Fontan circulation. They occur especially when central venous pressures are high, and very likely indicate arterialisation of hepatic blood flow and reciprocal portal venous deprivation. The underlying pathology is most likely focal nodular hyperplasia.

JunD is a profibrogenic transcription factor regulated by Jun N-terminal kinase-independent phosphorylation

JunD is implicated in the regulation of hepatic stellate cell (HSC) activation and liver fibrosis via its transcriptional regulation of the tissue inhibitor of metalloproteinases‐1 (TIMP‐1) gene. In the present study we found in vivo evidence of a role for JunD in fibrogenesis. Expression of JunD was demonstrated in alpha‐SMA‐positive activated HSCs of fibrotic rodents and human livers. The junD−/− mice were protected from carbon tetrachloride–induced fibrosis. The livers of injured junD−/− mice displayed significantly reduced formation of fibrotic crosslinked collagen and a smaller number of alpha‐SMA‐positive HSCs compared with those of wild‐type (wt) mice. Hepatic TIMP‐1 mRNA expression in injured junD−/− mice was 78% lower and in culture activated junD−/− HSCs was 50%‐80% lower than that in wt mice. In examining the signal transduction mechanisms that regulate JunD‐dependent TIMP‐1 expression, we found a role for phosphorylation of the Ser100 residue of JunD but ruled out JNK as a mediator of this event, suggesting ERK1/2 is utilized. In conclusion, a signaling pathway for the development of fibrosis involves the regulation of TIMP‐1 expression by phosphorylated JunD. (HEPATOLOGY 2006;44:1432–1440.)

Organotypic liver culture in a fluid-air interface using slices of neonatal rat and adult human tissue—a model of fibrosis in vitro

INTRODUCTION Fibrosis is the common end stage of most liver disease but there is no effective treatment currently available. We hypothesised that if viability of liver tissue slice culture could be improved, it should be possible to develop a model of liver fibrosis in vitro that could advance the development of antifibrotic therapy while at the same time reducing the need to use in vivo models. We have adapted a slice culture technique developed originally for organotypic culture of neural tissue to the liver. METHODS slices of neonatal rat or adult human liver, 100-400-microm thick, were cut and cultured on nitrocellulose inserts at the air/fluid interface for up to 28 days. RESULTS Hepatocytes expressed albumin by immunocytochemistry for up to 10 days and were viable for up to 21 days during which time new structures appeared, including cytokeratin 19 positive bile ductular structures and bands of smooth muscle actin positive stellate cells associated with new reticulin positive matrix. Smooth muscle actin expression by stellate cells could be pharmacologically inhibited by SDZ-RAD (everolimus). DISCUSSION In conclusion, we have successfully developed a novel model of liver culture, which may prove useful in both studies of the mechanisms of liver fibrosis and in developing therapeutic strategies.