Investigation of the role of hepatic stellate cells in acute liver failure and hepatocarcinogenesis

Abstract

Introduction: Hepatic stellate cells (HSC) and myofibroblasts may be relevant stromal drivers of human hepatocellular carcinoma (HCC). It was hypothesised that targeted inhibition of αv integrin-mediated TGF-β activation, by HSC or hepatocytes, may result in reduced peri-tumoural and intra-tumoural extracellular matrix formation, and reduced hepatic carcinogenesis. The role of HSC in acute liver injury is less well characterised. It was anticipated that integrin signalling on HSC and hepatocytes might also be relevant in the acute setting. The emerging technique of intravital microscopy (IVM) allows detailed, real-time investigation of the cellular processes involved in hepatocyte injury, cell death and repair. It was hypothesised that this could be coupled with mouse models of HCC and acute liver injury, to perform sequential imaging under anaesthesia. Aims: (i) To determine the effect of targeted inhibition of αv integrins on HSC and hepatocytes, during hepatocarcinogenesis, in a mouse model of HCC. (ii) To investigate the effect of targeted inhibition of αv and other integrins on HSC, hepatocytes, and liver sinusoidal endothelial cells (LSEC), during acute liver injury, in the mouse model of paracetamol-induced liver injury. (iii) To develop IVM of the liver, via an abdominal imaging window, with optimisation of surgical and imaging techniques, to allow sequential imaging of the same animal. Methods: The diethylnitrosamine (DEN)-induced mouse model of hepatocarcinogenesis was used, and PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were employed to deplete αv integrins on HSC and hepatocytes respectively. Tumours were harvested at 40 weeks post-DEN. Tumour size and number was evaluated in all animals. PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were used in the paracetamol model, to investigate the role of αv integrins in acute liver injury. PDGFRβ-Cre;β8fl/fl and Alb-Cre;β 8fl/fl animals were also tested in this model. The role of integrins in liver sinusoidal endothelial cells (LSEC) during paracetamol-induced liver injury was evaluated using Cdh5-Cre mice. IVM of the liver was performed by surgical implantation of an abdominal imaging window, consisting of a titanium ring and coverslip, secured in place with a purse string suture. Fluorescent reporter mice were used to identify hepatic and vascular architecture, and other label-free microscope technologies were utilised to image collagen, lipid distribution, necrotic areas and blood flow within tissues. Results: In large cohorts of PDGFRβ-Cre;αvfl/fl, Alb-Cre;αvfl/fl, and control animals, there was no difference in mean tumour size or number, at 40 weeks. Targeted inhibition of α v integrins and β 8 integrin on hepatocytes, HSC or LSEC was not protective in paracetamol-induced liver injury. IVM of the liver can be performed on animals with HCC and throughout paracetamol-induced liver injury, to obtain high quality, real-time images of multiple cell lineages and the hepatic microenvironment. Conclusions: The role of TGF-β in HCC pathogenesis is complex and context-dependent. Targeted loss of αv integrin did not result in reduction in tumour burden in this non-cirrhotic model of HCC. IVM of the liver is a powerful tool to quantify inflammatory infiltrates and assessment of vascular remodelling throughout the course of acute liver injury and regeneration, providing insights into the biological processes determining recovery.