Hepatic sinusoidal cell cross-talk is key to liver fibrosis resolution

Abstract

Background & Aims: Liver fibrosis is a wound-healing response to acute and chronic cellular injury of the liver. In the latter, normal parenchyma is replaced by scar tissue and eventually cirrhosis will develop. The burden of chronic liver disease is evident but unfortunately, no adequate therapies for liver fibrosis are available. Latest research has uncovered that inactivation of Hepatic Stellate Cells (HSCs) plays a major role in fibrosis regression and has emphasized on the importance of the liver micro-environment in fibrosis progression and regression. To achieve novel therapeutic targets, a more profound understanding of this micro-environment and the role of HSCs in fibrosis progression and regression is required. In this master dissertation, two goals were set to achieve this: the analysis of HSC–Liver sinusoidal endothelial cell (LSEC) crosstalk in vivo and the generation of a novel 3D coculture model to investigate HSC inactivation in vitro. Methods: For the in vivo part, BALB/c mice were treated with carbon tetrachloride (CCl4), inducing both acute and chronic injury with inclusion of several recovery groups. Livers of mice were collected for histological assessment and reverse transcription and real-time polymerase chain reaction were performed for analysis on RNA level. In the in vitro section, two types of 3D mouse liver spheroids were generated: Hepatocyte-HSC di-cultures and Hepatocyte-HSC-LSEC-Kupffer Cell quatro-cultures. These spheroids were incubated with the pro-mitotic and pro-fibrotic compounds, PDGF BB and TGF-β. Comparison between treated and control spheroids was achieved by RNA (reverse transcription and real-time polymerase chain reaction) and immunofluorescent assessment of HSC activation and mitosis. Results: Fibrosis resolution followed CCl4 withdrawal in the chronic injury group along with HSC inactivation as was proven by a decreased collagen deposition and upregulation of HSC inactivation markers. The lack of resolution in the acute injury group, was accompanied by a difference in the presence of Kupffer cells. Spheroids were cultured and kept their included cell types for the entire duration of the culture (4 days). Both the di-cultures and quatro-cultures showed properties of compound dependent activation and proliferation. However, the quatro-cultures seemed to have high activation and proliferation in control spheroids as well, indicating a potential role for Kupffer Cells or LSECs. Conclusion: In this master dissertation, the first steps were made to achieve new insights in fibrosis progression and reversal. First, a reversible fibrosis model was validated that will allow for more thorough in vivo analysis of the HSC-LSEC crosstalk and second, compound responsive 3D cultures were developed for further examination of inactivation in vitro.

Date of Award	Jun 2017
Original language	English
Supervisor	Inge Mannaerts (Co-promotor) & Hendrik Reynaert (Promotor)