Oihane García-Irigoyen

Enoxaparin reduces hepatic vascular resistance and portal pressure in cirrhotic rats

BACKGROUND & AIMS Increased hepatic vascular resistance due to fibrosis and elevated hepatic vascular tone is the primary factor in the development of portal hypertension. Heparin may decrease fibrosis by inhibiting intrahepatic microthrombosis and thrombin-mediated hepatic stellate cell activation. In addition, heparin enhances eNOS activity, which may reduce hepatic vascular tone. Our study aimed at evaluating the effects of acute, short-, long-term and preventive enoxaparin administration on hepatic and systemic hemodynamics, liver fibrosis and nitric oxide availability in cirrhotic rats. METHODS Enoxaparin (1.8 mg/kg subcutaneously), or its vehicle, was administered to CCl4-cirrhotic rats 24h and 1h before the study (acute), daily for 1 week (short-term) or daily for 3 weeks (long-term) and to thioacetamide-cirrhotic rats daily for 3 weeks with/without thioacetamide (preventive/long-term, respectively). Mean arterial pressure, portal pressure, portal blood flow, hepatic vascular resistance and molecular/cellular mechanisms were evaluated. RESULTS No significant changes in hemodynamic parameters were observed in acute administration. However, one-week, three-week and preventive treatments significantly decreased portal pressure mainly due to a decrease in hepatic vascular resistance without significant changes in mean arterial pressure. These findings were associated with significant reductions in liver fibrosis, hepatic stellate cell activation, and desmin expression. Moreover, a reduction in fibrin deposition was observed in enoxaparin-treated rats, suggesting reduced intrahepatic microthrombosis. CONCLUSION Enoxaparin reduces portal pressure in cirrhotic rats by improving the structural component of increased liver resistance. These findings describe the potentially beneficial effects of enoxaparin beyond the treatment/prevention of portal vein thrombosis in cirrhosis, which deserve further investigation.

Epidermal Growth Factor Receptor Signaling in Hepatocellular Carcinoma: Inflammatory Activation and a New Intracellular Regulatory Mechanism

Background/Aims: Hepatocellular carcinoma (HCC) is a chemoresistant tumor strongly associated with chronic hepatitis. Identification of molecular links connecting inflammation with cell growth/survival, and characterization of pro-tumorigenic intracellular pathways is therefore of therapeutic interest. The epidermal growth factor receptor (EGFR) signaling system stands at a crossroad between inflammatory signals and intracellular pathways associated with hepatocarcinogenesis. We investigated the regulation and activity of different components of the EGFR system, including the EGFR ligand amphiregulin (AR) and its sheddase ADAM17, and the modulation of intracellular EGFR signaling by a novel mechanism involving protein methylation. Methods: ADAM17 protein expression was examined in models of liver injury and carcinogenesis. Crosstalk between tumor necrosis factor (TNF)-α, AR and EGFR signaling was evaluated in human HCC cells and mouse hepatocytes. Modulation of EGFR signaling and biological responses by methylation reactions was evaluated in AML12 mouse hepatocytes. Results: ADAM17 was upregulated in liver injury and hepatocarcinogenesis. TNF-α triggered AR shedding and EGFR transactivation in HCC cells. AR was necessary for TNF-α activation of ERK1/2 and Akt signaling in hepatocytes. Inhibition of methylation reactions increased the ERK1/2 signal amplitude triggered by AR/EGFR and reduced DNA synthesis in AML12 cells. Conclusions: Increased ADAM17 in pre-neoplastic liver injury further supports its implication in hepatocarcinogenesis. AR release and EGFR transactivation by TNF-α constitutes a novel link between inflammatory signals and pro-tumorigenic mechanisms in liver cells. Finally, the identification of a new mechanism controlling growth factor signaling, and biological responses, involving methylation reactions within the RAS/RAF/MEK/ERK pathway, exposes a new target for antineoplastic intervention.

Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer

Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a “signaling hub” where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment.

Matrix metalloproteinase‐10 expression is induced during hepatic injury and plays a fundamental role in liver tissue repair

Upon tissue injury, the liver mounts a potent reparative and regenerative response. A role for proteases, including serine and matrix metalloproteinases (MMPs), in this process is increasingly recognized. We have evaluated the expression and function of MMP10 (stromelysin‐2) in liver wound healing and regeneration.

Matrix metalloproteinase 10 contributes to hepatocarcinogenesis in a novel crosstalk with the stromal derived factor 1/C‐X‐C chemokine receptor 4 axis

Matrix metalloproteinases (MMPs) participate in tissue repair after acute injury, but also participate in cancer by promoting a protumorigenic microenvironment. Previously, we reported on a key role for MMP10 in mouse liver regeneration. Herein, we investigated MMP10 expression and function in human hepatocellular carcinoma (HCC) and diethylnitrosamine (DEN)‐induced mouse hepatocarcinogenesis. MMP10 was induced in human and murine HCC tissues and cells. MMP10‐deficient mice showed less HCC incidence, smaller histological lesions, reduced tumor vascularization, and less lung metastases. Importantly, expression of the protumorigenic, C‐X‐C chemokine receptor‐4 (CXCR4), was reduced in DEN‐induced MMP10‐deficient mice livers. Human HCC cells stably expressing MMP10 had increased CXCR4 expression and migratory capacity. Pharmacological inhibition of CXCR4 significantly reduced MMP10‐stimulated HCC cell migration. Furthermore, MMP10 expression in HCC cells was induced by hypoxia and the CXCR4 ligand, stromal‐derived factor‐1 (SDF1), through the extracellular signal‐regulated kinase 1/2 pathway, involving an activator protein 1 site in MMP10 gene promoter. Conclusion: MMP10 contributes to HCC development, participating in tumor angiogenesis, growth, and dissemination. We identified a new reciprocal crosstalk between MMP10 and the CXCR4/SDF1 axis contributing to HCC progression and metastasis. To our knowledge, this is the first report addressing the role of a MMP in hepatocarcinogenesis in the corresponding genetic mouse model. (Hepatology 2015;62:166‐178)

Regulation of amphiregulin gene expression by β-catenin signaling in human hepatocellular carcinoma cells: a novel crosstalk between FGF19 and the EGFR system.

Hepatocellular carcinoma (HCC) is the most prevalent liver tumor and a deadly disease with limited therapeutic options. Dysregulation of cell signaling pathways is a common denominator in tumorigenesis, including hepatocarcinogenesis. The epidermal growth factor receptor (EGFR) signaling system is commonly activated in HCC, and is currently being evaluated as a therapeutic target in combination therapies. We and others have identified a central role for the EGFR ligand amphiregulin (AR) in the proliferation, survival and drug resistance of HCC cells. AR expression is frequently up-regulated in HCC tissues and cells through mechanisms not completely known. Here we identify the β-catenin signaling pathway as a novel mechanism leading to transcriptional activation of the AR gene in human HCC cells. Activation of β-catenin signaling, or expression of the T41A β-catenin active mutant, led to the induction of AR expression involving three specific β-catenin-Tcf responsive elements in its proximal promoter. We demonstrate that HCC cells expressing the T41A β-catenin active mutant show enhanced proliferation that is dependent in part on AR expression and EGFR signaling. We also demonstrate here a novel cross-talk of the EGFR system with fibroblast growth factor 19 (FGF19). FGF19 is a recently identified driver gene in hepatocarcinogenesis and an activator of β-catenin signaling in HCC and colon cancer cells. We show that FGF19 induced AR gene expression through the β-catenin pathway in human HCC cells. Importantly, AR up-regulation and EGFR signaling participated in the induction of cyclin D1 and cell proliferation elicited by FGF19. Finally, we demonstrate a positive correlation between FGF19 and AR expression in human HCC tissues, therefore supporting in clinical samples our experimental observations. These findings identify the AR/EGFR system as a key mediator of FGF19 responses in HCC cells involving β-catenin signaling, and suggest that combined targeting of FGF19 and AR/EGFR may enhance therapeutic efficacy.

Splicing regulator SLU7 is essential for maintaining liver homeostasis

A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.

The anticoagulant Rivaroxaban lowers portal hypertension in cirrhotic rats mainly by deactivating hepatic stellate cells

In cirrhosis, increased intrahepatic vascular resistance (IHVR) is the primary factor for portal hypertension (PH) development. Hepatic stellate cells (HSCs) play a major role increasing IHVR because, when activated, they are contractile and promote fibrogenesis. Protease‐activated receptors (PARs) can activate HSCs through thrombin and factor Xa, which are known PAR agonists, and cause microthrombosis in liver microcirculation. This study investigates the effects of the oral anticoagulant, rivaroxaban (RVXB), a direct antifactor Xa, on HSC phenotype, liver fibrosis (LF), liver microthrombosis, and PH in cirrhotic rats. Hepatic and systemic hemodynamic, nitric oxide (NO) bioavailability, LF, HSC activation, and microthrombosis were evaluated in CCl4 and thioacetamide‐cirrhotic rats treated with RVXB (20 mg/kg/day) or its vehicle for 2 weeks. RVXB significantly decreased portal pressure (PP) in both models of cirrhosis without changes in portal blood flow, suggesting a reduction in IHVR. RVXB reduced oxidative stress, improved NO bioavailability, and ameliorated endothelial dysfunction. Rivaroxaban deactivated HSC, with decreased alpha‐smooth muscle actin and mRNA expression of other HSC activation markers. Despite this marked improvement in HSC phenotype, no significant changes in LF were identified. RVXB markedly reduced fibrin deposition, suggesting reduced intrahepatic microthrombosis. Conclusion: RVXB decreases PP in two rat models of cirrhosis. This effect is mostly associated with decreased IHVR, enhanced NO bioavailability, HSC deactivation, and reduced intrahepatic microthrombosis. Our findings suggest that RVXB deserves further evaluation as a potential treatment for cirrhotic PH. (Hepatology 2017;65:2031‐2044).

Alterations in the expression and activity of pre-mRNA splicing factors in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is a molecularly complex tumor that is resistant to standard and targeted therapies, and thus a deadly disease. In this context, the identification of key alterations driving HCC development is therefore essential. The implementation of next-generation sequencing techniques has underscored earlier realizations of the marked dysregulation of pre-mRNA splicing in HCC. Impairments in alternative splicing may lead to the expression of protumorigenic protein isoforms and to the generation of unstable mRNA species. Mechanistically, mutations in key nucleotides are responsible for many of these alterations in different types of tumors. However, changes in the expression of factors involved in the regulation of the splicing machinery are also important determinants in the derangement of pre-mRNA splicing. Here we discuss recent reports on the alteration of splicing factors in HCC, the pathological significance of these changes, and the identification of cell signaling pathways leading to the missplicing of genes in hepatocarcinogenesis.

Hepatic matrix metalloproteinase-10 exerts a hepatoprotective role after acute liver injury

After injuries that lead to a loss of liver tissue a regenerative and reparative response is performed in order to restore an adequate hepatic mass. The remodeling of the extracellular matrix, accompanies the liver regeneration and when the reparative reaction goes awry in the setting of chronic liver injury, could be involved in the carcinogenic process (1,2). Following the damage, a provisional matrix is deposed, intended to be successively replaced, which has the function of stabilizing the lesional area and constitutes a support for guiding regenerating cells. Matrix metalloproteinases are increasingly recognized as important modulators of the matrix remodeling process. Matrix metalloproteinase-10 (MMP-10) has been implicated in the reparative process in other organs and has effects on the plasminogen system, which plays a fundamental role in liver repair (3). The hepatic expression of MMP10 in animal models of acute liver injury was tested in order to investigate the role of MMP-10 in liver repair and regeneration. The liver regeneration after two thirds partial hepatectomy (PH) and bile duct ligation (BDL) models were examined. Hepatic MMP-10 expression, analyzed by immunohistochemistry, western blot and qPCR showed a rise early after injury. In the MMP10-deficient mice a diminished and delayed resolution of necrotic lesions, enhanced fibrogenesis and a fibrinogen/fibrin and fibronectin compromised turnover were observed. These findings showed that the MMP10 expression plays a role in the hepatic wound healing response probably through its profibrinolytic activity.