Disrupted bile secretion leads to liver damage characterized by inflammation, fibrosis, eventually cirrhosis, and hepatocellular cancer. As obstructive cholestasis often progresses insidiously, markers for the diagnosis and staging of the disease are urgently needed. To this end, we compiled a comprehensive data set of serum markers, histological parameters and transcript profiles at 8 time points of disease progression after bile duct ligation (BDL) in mice, aiming at identifying a set of parameters that could be used as robust biomarkers for transition of different disease progression phases. Statistical analysis of the more than 6,000 data points revealed distinct temporal phases of disease. Time course correlation analysis of biochemical, histochemical and mRNA transcript parameters (=factors) defined 6 clusters for different phases of disease progression. The number of CTGF-positive cells provided the most reliable overall measure for disease progression at histological level, bilirubin at biochemical level, and metalloproteinase inhibitor 1 (Timp1) at transcript level. Prominent molecular events exhibited by strong transcript peaks are found for the transcriptional regulator Nr0b2 (Shp) and 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1) at 6 h. Based on these clusters, we constructed a decision tree of factor combinations potentially useful as markers for different time intervals of disease progression. Best prediction for onset of disease is achieved by fibronectin (Fn1), for early disease phase by Cytochrome P450 1A2 (Cyp1a2), passage to perpetuation phase by collagen1α-1 (Col1a1), and transition to the progression phase by interleukin 17-a (Il17a), with early and late progression separated by Col1a1. Notably, these predictions remained stable even for randomly chosen small sub-sets of factors selected from the clusters. Our detailed time-resolved explorative study of liver homogenates following BDL revealed a well-coordinated response, resulting in disease phase dependent parameter modulations at morphological, biochemical, metabolic and gene expression levels. Interestingly, a small set of selected parameters can be used as diagnostic markers to predict disease stages in mice with cholestatic liver disease.

Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGF_p) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGF_p to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGF_p to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGF_p transduce activated HSCs better than Ad.GFP-PEG-NGF_p. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGF_p.

Liver resection and the use of small-for-size grafts are restricted by the necessity to provide a sufficient amount of functional liver mass. Only few promising strategies to maximize liver regeneration are available. Apart from its erythropoiesis-stimulating effect, erythropoietin (EPO) has meanwhile been recognized as mitogenic, tissue-protective, and anti-apoptotic pleiotropic cytokine. Thus, EPO may support regeneration of hepatic tissue. Rats undergoing 68% hepatectomy received daily either high dose (5000 IU/kg bw iv) or low dose (500 IU/kg bw iv) recombinant human EPO or equal amounts of physiologic saline. Parameters of liver regeneration and hepatocellular apoptosis were assessed at 24 h, 48 h and 5 d after resection. In addition, red blood cell count, hematocrit and serum EPO levels as well as plasma concentrations of TNF-α and IL-6 were evaluated. Further, hepatic Bcl-x_L and Bax protein expression were analyzed by Western blot. Administration of EPO significantly reduced the expression of PCNA at 24 h followed by a significant decrease in restitution of liver mass at day 5 after partial hepatectomy. EPO increased TNF-α levels and shifted the Bcl-x_L to Bax ratio towards the pro-apoptotic Bax resulting in significantly increased hepatocellular apoptosis. Multiple doses of EPO after partial hepatectomy increase hepatocellular apoptosis and impair liver regeneration in rats. Thus, careful consideration should be made in pre- and post-operative recombinant human EPO administration in the setting of liver resection and transplantation.

Background: With 9.1% of all cancer deaths, hepatocellular carcinoma is the second leading cause of cancer deaths worldwide. Due to the increasing prevalence of metabolic syndrome, nonalcoholic fatty liver disease (NAFLD) has evolved into a major risk factor for hepatocellular carcinoma development. Herein, we investigated whether a dietary n-3 polyunsaturated fatty acid (PUFA) supplementation improves the outcome of progressive NAFLD. Methods: Feeding three high-fat diets, differing in n-3 and n-6 PUFA contents and ratios (n-3/n-6: 1:8, 1:1, 5:1), the impact of n-3 PUFAs and n-3/n-6 PUFA ratios on NAFLD-related liver fibrosis and tumorigenesis was analyzed in 12- and 20-week-old streptozotocin/high-fat diet (STZ/HFD)-treated mice. Results: Feeding of n-3 PUFA-rich diets (1:1 and 5:1) resulted in increased hepatic n-3 PUFA content and n-3/n-6 PUFA ratio with decreased hepatic lipid accumulation. In 20-week-old mice, n-3 PUFA-rich diets alleviated tumor load significantly, with reduced liver/body weight index, tumor size, and tumor number. Finally, these effects were accompanied by a significant improvement of survival of these mice. Conclusions: Herein, we showed that increased n-3 PUFA content and n-3/n-6 PUFA ratios lead to improved survival and attenuated tumor progression in STZ/HFD-treated mice. Thus, n-3 PUFAs could be the basis for new therapeutic options against NAFLD-related tumorigenesis.