Gianluca Svegliati Baroni

Molecular basis and mechanisms of progression of non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH), a cause of cirrhosis and hepatocellular carcinoma, is characterized by fatty infiltration of the liver, inflammation, hepatocellular damage and fibrosis. Progress has been made in understanding the molecular and cellular mechanisms implicated in the pathogenesis of this condition, therefore, we here review recent developments regarding the basic mechanisms of NASH development. Accumulation of triglycerides in the hepatocytes is the result of increased inflow of free fatty acids and de novo lipogenesis. Steatosis leads to lipotoxicity, which causes apoptosis, necrosis, generation of oxidative stress and inflammation. The resulting chronic injury activates a fibrogenic response that leads eventually to end-stage liver disease. A better understanding of these mechanisms is crucial for the design of novel diagnostic and therapeutic strategies.

Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans

Epithelial-mesenchymal transitions (EMTs) play an important role in tissue construction during embryogenesis, and evidence suggests that this process may also help to remodel some adult tissues after injury. Activation of the hedgehog (Hh) signaling pathway regulates EMT during development. This pathway is also induced by chronic biliary injury, a condition in which EMT has been suggested to have a role. We evaluated the hypothesis that Hh signaling promotes EMT in adult bile ductular cells (cholangiocytes). In liver sections from patients with chronic biliary injury and in primary cholangiocytes isolated from rats that had undergone bile duct ligation (BDL), an experimental model of biliary fibrosis, EMT was localized to cholangiocytes with Hh pathway activity. Relief of ductal obstruction in BDL rats reduced Hh pathway activity, EMT, and biliary fibrosis. In mouse cholangiocytes, coculture with myofibroblastic hepatic stellate cells, a source of soluble Hh ligands, promoted EMT and cell migration. Addition of Hh-neutralizing antibodies to cocultures blocked these effects. Finally, we found that EMT responses to BDL were enhanced in patched-deficient mice, which display excessive activation of the Hh pathway. Together, these data suggest that activation of Hh signaling promotes EMT and contributes to the evolution of biliary fibrosis during chronic cholestasis.

Effect of pirfenidone on rat hepatic stellate cell proliferation and collagen production

BACKGROUND/AIMS Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC). METHODS HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively. RESULTS Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression. RESULTS Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

Chronic ethanol feeding increases apoptosis and cell proliferation in rat liver

The present study was conducted to evaluate if the increased rate of apoptosis previously reported in the liver of ethanol-treated rats was accompanied by increased cell renewal. A quantitative analysis of apoptosis was performed in rats fed an ethanol-containing liquid diet for 5 weeks. S-phase cells were demonstrated by immunohistochemistry, using the Bromodeoxyuridine/anti-Bromodeoxyuridine method. In ethanol-fed rats apoptosis was five times greater than in pair-fed controls. Bromodeoxyuridine-labelled hepatocytes increased from 0.07 +/- 0.009% in controls to 0.17 +/- 0.013% (p < 0.001) and Bromodeoxyuridine-labelled lipocytes (desmin-positive sinusoidal cells) increased from 3.43 +/- 0.28% to 6.60 +/- 1.04% (p < 0.001). The lobular distribution of labelled cells was modified with a shift towards the perivenular areas. The results of this study suggest that the replacement of liver cells lost by ethanol-induced apoptosis is not impaired in intact (non-operated) animals. The impaired regeneration following partial hepatectomy reported in ethanol-fed rats is possibly due to differences in the extent of parenchymal loss, to altered relationships between hepatocytes and blood supply and to the modalities of regeneration involved.

Practice guidelines for the treatment of hepatitis C: Recommendations from an AISF/SIMIT/SIMAST expert opinion meeting

It is increasingly clear that a tailored therapeutic approach to patients with hepatitis C virus infection is needed. Success rates in difficult to treat and low-responsive hepatitis C virus patients are not completely satisfactory, and there is the need to optimise treatment duration and intensity in patients with the highest likelihood of response. In addition, the management of special patient categories originally excluded from phase III registration trials needs to be critically re-evaluated. This article reports the recommendations for the treatment of hepatitis C virus infection on an individual basis, drafted by experts of three scientific societies.

Intracellular pathways mediating Na+/H+ exchange activation by platelet-derived growth factor in rat hepatic stellate cells

BACKGROUND & AIMS The Na+/H+ exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na+/H+ exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na+/H+ exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na+/H+ exchanger by PDGF in HSCs. METHODS The activity of the Na+/H+ exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na+/H+ exchange protein expression was evaluated by means of Western blot. RESULTS PDGF induced a significant increase in the activity of the Na+/H+ exchanger without modifying protein expression. Inhibition of the calcium/calmodulin- and protein kinase C-dependent pathways resulted in a significant inhibition of both Na+/H+ exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na+/H+ exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na+/H+ exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase- and of phosphatidylinositol 3-kinase-dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na+/H+ exchanger. CONCLUSIONS Activation of the Na+/H+ exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C-dependent pathways. PDGF-induced HSC proliferation is mediated by Na+/H+ exchange-dependent and -independent pathways.

Estimation of lead-time bias and its impact on the outcome of surveillance for the early diagnosis of hepatocellular carcinoma

BACKGROUND & AIMS Lead-time is the time by which diagnosis is anticipated by screening/surveillance with respect to the symptomatic detection of a disease. Any screening program, including surveillance for hepatocellular carcinoma (HCC), is subject to lead-time bias. Data regarding lead-time for HCC are lacking. Aims of the present study were to calculate lead-time and to assess its impact on the benefit obtainable from the surveillance of cirrhotic patients. METHODS One-thousand three-hundred and eighty Child-Pugh class A/B patients from the ITA.LI.CA database, in whom HCC was detected during semiannual surveillance (n = 850), annual surveillance (n = 234) or when patients came when symptomatic (n = 296), were selected. Lead-time was estimated by means of appropriate formulas and Monte Carlo simulation, including 1000 patients for each arm. RESULTS The 5-year overall survival after HCC diagnosis was 32.7% in semiannually surveilled patients, 25.2% in annually surveilled patients, and 12.2% in symptomatic patients (p<0.001). In a 10-year follow-up perspective, the median lead-time calculated for all surveilled patients was 6.5 months (7.2 for semiannual and 4.1 for annual surveillance). Lead-time bias accounted for most of the surveillance benefit until the third year of follow-up after HCC diagnosis. However, even after lead-time adjustment, semiannual surveillance maintained a survival benefit over symptomatic diagnosis (number of patients needed to screen = 13), as did annual surveillance (18 patients). CONCLUSIONS Lead-time bias is the main determinant of the short-term benefit provided by surveillance for HCC, but this benefit becomes factual in a long-term perspective, confirming the clinical utility of an anticipated diagnosis of HCC.

Survival benefit of liver resection for patients with hepatocellular carcinoma across different Barcelona Clinic Liver Cancer stages: A multicentre study

BACKGROUND & AIMS The role of hepatic resection for hepatocellular carcinoma (HCC) in different Barcelona Clinic Liver Cancer (BCLC) stages is controversial. We aimed at measuring the survival benefit of resection vs. non-surgical-therapies in each BCLC stage. METHODS Using the ITA.LI.CA database, we identified 2090 BCLC A, B, and C HCC patients observed between 2000 and 2012: 550 underwent resection, 1046 loco-regional therapy (LRT), and 494 best supportive care (BSC). A multivariate log-logistic model was chosen to predict median survival (MS) after resection vs. MS after LRT or BSC. The results were expressed as net survival benefit of resection: (MS resection-MS LRT)/MS BSC. RESULTS After stratifying for BCLC stage, the median net survival benefit of resection over LRT was: BCLC 0=62% (40%, 82%), A=45% (13%, 65%), B=46% (9%, 76%), C=-16% (-55%, 33%). Model for end-stage liver disease (MELD) score>9, Child B class, and performance status (PST)=2 were the main risk factors for liver resection. 1181 Child A patients (57%) with MELD⩽9 and PST<2 had always a large positive net survival benefit of resection over LRT, independently of BCLC stage: BCLC 0=64% (44%, 85%), A=59% (45%, 74%), B=71% (52%, 90%), C=56% (36%, 78%). Among the 909 (43%) patients with at least one risk factor (MELD>9 or PST=2 or Child B class), resection did not prove any survival benefit over LRT. CONCLUSIONS Resection could result in survival benefit over LRT for HCC patients regardless of their BCLC stage, provided that liver dysfunction (Child B or MELD>9) and PST>1 are absent.

Intracellular pH regulation and Na+/H+ exchange activity in human hepatic stellate cells: effect of platelet-derived growth factor, insulin-like growth factor 1 and insulin

BACKGROUND/AIMS The Na+/H+ exchanger is involved in rat hepatic stellate cell (HSC) proliferation induced by platelet-derived growth factor (PDGF). We therefore evaluated in human HSC: (1) the mechanisms of intracellular pH regulation; (2) the relationship between Na+/H+ exchange activation and cell proliferation induced by PDGF, insulin-like growth factor 1 (IGF-1) and insulin. METHODS/RESULTS pH(i) regulation was mainly dependent on the activity of the Na+/H+ exchanger, which was evaluated by measuring pH(i) recovery from an acute acid load. PDGF (25 ng/ml) gradually increased the activity of the Na+/H+ exchanger which peaked at 18 h and remained stable until the 24th h. IGF-1 (10 nmol/l), but not insulin (100 nmol/l), slightly but significantly increased the activity of the Na+/H+ exchanger. Amiloride (100 micromol/l) and 20 micromol/l 5-N-ethyl-N-isopropyl-amiloride completely inhibited HSC proliferation (evaluated by measurement of bromodeoxyuridine incorporation) induced by PDGF and IGF-1, but did not affect proliferation of HSC induced by insulin. Finally, IGF-1 did not modify the activity of the Na+/Ca2+ exchanger. CONCLUSIONS The Na+/H+ exchanger is involved in HSC proliferation induced by PDGF and IGF-1, whereas the proliferative effect of insulin is mediated by intracellular pathways which are Na+/H+ exchange-independent.

The Role of LDH Serum Levels in Predicting Global Outcome in HCC Patients Undergoing TACE: Implications for Clinical Management

In many tumor types serum lactate dehydrogenase (LDH) levels is an indirect marker of tumor hypoxia, neo-angiogenesis and worse prognosis. However data about hepatocellular carcinoma (HCC) are lacking in the clinical setting of patients undergoing transarterial-chemoembolization (TACE) in whom hypoxia and neo-angiogenesis may represent a molecular key to treatment failure. Aim of our analysis was to evaluate the role of LDH pre-treatment levels in determining clinical outcome for patients with HCC receiving TACE. One hundred and fourteen patients were available for our analysis. For all patients LDH values were collected within one month before the procedure. We divided our patients into two groups, according to LDH serum concentration registered before TACE (first: LDH≤450 U/l 84 patients; second: LDH>450 U/l 30 patients). Patients were classified according to the variation in LDH serum levels pre- and post-treatment (increased: 62 patients vs. decreased 52 patients). No statistically significant differences were found between the groups for all clinical characteristics analyzed (gender, median age, performance status ECOG, staging systems). In patients with LDH values below 450 U/l median time to progression (TTP) was 16.3 months, whereas it was of 10.1 months in patients above the cut-off (p = 0.0085). Accordingly median overall survival (OS) was 22.4 months and 11.7 months (p = 0.0049). In patients with decreased LDH values after treatment median TTP was 12.4 months, and median OS was 22.1 months, whereas TTP was 9.1 months and OS was 9.5 in patients with increased LDH levels (TTP: p = 0.0087; OS: p<0.0001). In our experience, LDH seemed able to predict clinical outcome for HCC patients undergoing TACE. Given the correlation between LDH levels and tumor angiogenesis we can speculate that patients with high LDH pretreatment levels may be optimal candidates for clinical trial exploring a multimodality treatment approach with TACE and anti-VEGF inhibitors in order to improve TTP and OS.