Laura Conde de la Rosa

Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine‐conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)‐induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco‐conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol‐3 kinase pathway (PI3K) and nuclear factor‐κB were inhibited using LY 294002 and adenoviral overexpression of dominant‐negative IκB, respectively. The role of p38 and extracellular signal‐regulated protein kinase mitogen‐activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin‐D. Apoptosis was determined by measuring caspase‐3, ‐9, and ‐8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase‐8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA‐induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF‐κB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA‐induced mitochondria‐controlled apoptosis by activating survival pathways. Supplemental material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/supplmat/index.html). (HEPATOLOGY 2004;39:1563–1573.)

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation

BACKGROUND/AIMS To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis. METHODS Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3. RESULTS Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2. CONCLUSIONS GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.

Contrast-Enhanced Versus Conventional and Color Doppler Sonography for the Detection of Thrombosis of the Portal and Hepatic Venous Systems

OBJECTIVE We conducted a prospective study to compare sonography, color Doppler sonography, and contrast-enhanced sonography for the detection and characterization of portal and hepatic vein thrombosis complicating hepatic malignancies. SUBJECTS AND METHODS Three hundred sixteen patients with biopsy-proved hepatic tumors were studied at baseline and 3 months later with sonography, color Doppler sonography, and contrast-enhanced sonography. Thrombosis was defined as the presence of intraluminal echogenic material at sonography, absence of intraluminal color signals at color Doppler sonography, and presence of nonenhancing intraluminal area at contrast-enhanced sonography. Thrombi were considered malignant if they displayed continuity with tumor tissue at sonography, intrathrombus color signals at color Doppler sonography, and enhancing signals at contrast-enhanced sonography, both having arterial waveforms at Doppler spectral examination. Definitive diagnoses were obtained by sonographically guided biopsy except for thrombi displaying at conventional sonography unequivocal continuity with tumor tissue. RESULTS Thrombosis was detected in 79 (25.0%) of 316 patients at baseline and in 83 (26.3%) of 316 patients after 3 months. Eighty-one (97.6%) of the 83 thrombi were malignant. Definitive diagnosis was performed by imaging in 60 (72.3%) of the 83 cases and by biopsy in 23 cases (27.7%). For thrombus detection, contrast-enhanced sonography displayed significantly higher sensitivity than color Doppler sonography (p = 0.004) and borderline superiority over sonography (p = 0.058). For thrombus characterization, contrast-enhanced sonography was significantly more sensitive than color Doppler sonography (p < 0.0005) and conventional sonography (p = 0.02). CONCLUSION Contrast-enhanced sonography is superior to sonography and color Doppler sonography for the detection and characterization of portal and hepatic vein thrombosis complicating hepatic malignancies.

Repeated radiofrequency ablation for management of patients with cirrhosis with small hepatocellular carcinomas: A long‐term cohort study

In most patients with cirrhosis, successful percutaneous ablation or surgical resection of hepatocellular carcinoma (HCC) is followed by recurrence. Radiofrequency ablation (RFA) has proven effective for treating HCC nodules, but its repeatability in managing recurrences and the impact of this approach on survival has not been evaluated. To this end, we retrospectively analyzed a prospective series of 706 patients with cirrhosis (Child‐Pugh class ≤B7) who underwent RFA for 859 HCC ≤35 mm in diameter (1‐2 per patient). The results of RFA were classified as complete responses (CRs) or treatment failures. CRs were obtained in 849 nodules (98.8%) and 696 patients (98.5%). During follow‐up (median, 29 months), 465 (66.8%) of the 696 patients with CRs experienced a first recurrence at an incidence rate of 41 per 100 person‐years (local recurrence 6.2; nonlocal 35). Cumulative incidences of first recurrence at 3 and 5 years were 70.8% and 81.7%, respectively. RFA was repeated in 323 (69.4%) of the 465 patients with first recurrence, restoring disease‐free status in 318 (98.4%) cases. Subsequently, RFA was repeated in 147 (65.9%) of the 223 patients who developed a second recurrence after CR of the first, restoring disease‐free status in 145 (98.6%) cases. Overall, there were 877 episodes of recurrence (1‐8 per patient); 577 (65.8%) of these underwent RFA that achieved CRs in 557 (96.5%) cases. No procedure‐related deaths occurred in 1,921 RFA sessions. Estimated 3‐ and 5‐year overall and disease‐free (after repeated RFAs) survival rates were 67.0% and 40.1% and 68.0 and 38.0%, respectively. Conclusion: RFA is safe and effective for managing HCC in patients with cirrhosis, and its high repeatability makes it particularly valuable for controlling intrahepatic recurrences. (HEPATOLOGY 2011)

Contrast-enhanced ultrasonography and spiral computed tomography in the detection and characterization of portal vein thrombosis complicating hepatocellular carcinoma

The aim was to compare the performances of contrast-enhanced (CE) ultrasonography (US) and spiral computed tomography (CT) in the detection and characterization of portal vein thrombosis complicating hepatocellular carcinoma (HCC). We studied 50 patients with HCC who had biopsy-proven portal vein thrombi that had been detected with US and color Doppler US. Thirteen of the thrombi involved the main portal trunk and 37 the segmental branches. CEUS and CT were performed within a week of thrombus biopsies. For each imaging technique, diagnoses of thrombosis (present/absent) and thrombus nature (malignancy/benignancy) were made by experienced readers under blinded conditions and compared with pathological findings to determine accuracy rates for thrombus detection and characterization. Forty-four of the 50 thrombi were pathologically diagnosed as malignant and the remaining six were benign. CEUS detected 50/50 (100%) thrombi and correctly characterized 49/50 (98%). CT detected 34/50 (68%) thrombi and correctly characterized 23 of these 34 (68%). CEUS outperformed CT in terms of both thrombus detection (P < 0.0001) and characterization (P = 0.0001). CEUS appears to be significantly superior to CT for detection and characterization of portal vein thrombosis complicating HCC, and it should be considered in the staging of these tumors.

Glutamine synthetase immunostaining correlates with pathologic features of hepatocellular carcinoma and better survival after radiofrequency thermal ablation.

Purpose: Activation of the wnt pathway identifies a subgroup of hepatocellular carcinomas (HCC) with specific epidemiologic and genetic profiles. Wnt activation is predicted by mutation and/or nuclear translocation of β-catenin and by glutamine synthetase (GS) immunoreactivity. We investigated whether GS staining associates with specific pathologic features of HCC and with survival after radiofrequency thermal ablation. Experimental Design: Monoistitutional retrospective-prospective study in a tertiary hospital setting. Two hundred and seven cirrhotics (mean age, 70 years; 63% males, 82.1% hepatitis C virus positive) with early HCC were consecutively treated with radiofrequency thermal ablation (RFTA). Mean tumor size was 2.7 cm; 20.3% of patients had multiple nodules; and median follow-up was 36 months with 54.6% overall mortality. Tumor samples were mainly obtained by biopsy (92,5%) and examined by H&E and immunostaining for β-catenin and GS. Main outcome measures were overall and tumor-specific mortality by Kaplan-Meier analysis and Cox proportional hazard models corrected for competing risks. Results: Ninety-one patients (43.9%) had GS-positive HCCs by immunostaining. These tumors had larger size (P = 0.012) and characteristic histology (low grade, pseudoacini, hydropic changes, bile staining, lack of steatosis, and fibrosis). Other clinical or treatment variables were similar between groups. Variables correlating with tumor-specific and overall mortality by univariate analysis were tumor recurrence, advanced disease, posttreatment α-fetoprotein levels, and GS staining. Yearly, overall mortality rate was lower in GS-positive patients (12.4 versus 20% yearly; P = 0.006). By multivariate analysis, GS immunostaining correlated with reduced specific (hazard ratio, 0.58; 95% confidence interval, 0.34-0.97) and overall mortality (hazard ratio, 0.62; 95% confidence interval, 0.40-0.96). Conclusions: Standard histology and GS status identify a HCC subset with distinct clinical and pathologic features. Clin Cancer Res; 16(7); 2157–66. ©2010 AACR.

Patterns of appearance and risk of misdiagnosis of intrahepatic cholangiocarcinoma in cirrhosis at contrast enhanced ultrasound

Primary aim was to validate the percentage of intrahepatic cholangiocarcinomas (ICC) which have a contrast vascular pattern at contrast enhanced ultrasound (CEUS) at risk of misdiagnosis with hepatocellular carcinoma (HCC) and, secondary aim, to verify if any characteristics in the CEUS pattern helps to identify ICC.

Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform.

Most chronic liver diseases are accompanied by oxidative stress, which may induce apoptosis in hepatocytes and liver injury. Oxidative stress induces heme oxygenase-1 (HO-1) expression. This stress-responsive cytoprotective protein is responsible for heme degradation into carbon monoxide (CO), free iron, and biliverdin. CO is an important intracellular messenger; however, the exact mechanisms responsible for its cytoprotective effect are not yet elucidated. Thus, we investigated whether HO-1 and CO protect primary hepatocytes against oxidative-stress-induced apoptosis. In vivo, bile duct ligation was used as model of chronic liver disease. In vitro, primary hepatocytes were exposed to the superoxide anion donor menadione in a normal and in a CO-- containing atmosphere. Apoptosis was determined by measuring caspase-9, -6, -3 activity and poly(ADP-ribose) polymerase cleavage, and necrosis was determined by Sytox green staining. The results showed that (1) HO-1 is induced in chronic cholestatic liver disease, (2) superoxide anions time- and dose-dependently induce HO-1 activity, (3) HO-1 overexpression inhibits superoxide-anions-induced apoptosis, and (4) CO blocks superoxide-anions-induced JNK phosphorylation and caspase-9, -6, -3 activation and abolishes apoptosis but does not increase necrosis. We conclude that HO-1 and CO protect primary hepatocytes against superoxide-anions-induced apoptosis partially via inhibition of JNK activity. CO could represent an important candidate for the treatment of liver diseases.

Metformin protects primary rat hepatocytes against oxidative stress-induced apoptosis

The majority of chronic liver diseases are accompanied by oxidative stress, which induces apoptosis in hepatocytes and liver injury. Recent studies suggest that oxidative stress and insulin resistance are important in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the pathophysiology of diabetes complications. Metformin has been shown to be hepatoprotective in the insulin‐resistant and leptin‐deficient ob/ob mouse model of NAFLD. However, the mechanism involved in the protective effects of metformin has not been elucidated yet. Therefore, we investigated the protective effect of metformin against oxidative stress‐induced apoptosis. Primary rat hepatocytes were exposed to the oxidative stress‐generating compound menadione in the presence and absence of metformin. Apoptosis was determined by measuring caspase activity and poly(ADP‐ribose) polymerase (PARP)‐cleavage, and necrosis was measured by Sytox Green nuclear staining. We demonstrate that (1) Metformin inhibits menadione‐induced caspase‐9,‐6,‐3 activation and PARP‐cleavage in a concentration‐dependent manner. (2) Metformin increases menadione‐induced heme oxygenase‐1 (HO‐1) expression and inhibits c‐Jun N‐terminal kinase (JNK)‐phosphorylation. (3) Metformin does not induce necrosis in primary hepatocytes. Metformin protects hepatocytes against oxidative stress‐induced caspase activation, PARP‐cleavage and apoptosis. The anti‐apoptotic effect of metformin is in part dependent on HO‐1 and bcl‐xl induction and inhibition of JNK activation and independent of insulin signaling. Our results elucidate novel protective mechanisms of metformin and indicate that metformin could be investigated as a novel therapeutic agent for the treatment of oxidative stress‐related liver diseases.

Metformin protects rat hepatocytes against bile acid-induced apoptosis

Background Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death. Aim To evaluate the effects of metformin on hepatocyte cell death. Methods Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. Results Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. Conclusion Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.