The potential role of liver sinusoidal endothelial cells in drug-induced liver injury

Liver sinusoidal endothelial cells (LSEC) constitute a unique population of endothelial cells with specialised liver-specific morphologic features and functions. LSEC are the only endothelial cells with fenestrations and which lack an organised basement membrane. They are involved in hepatic stellate cell (HSC) quiescence, endocytosis of small particles, selective transfer of substances from the blood, in the hepatic sinusoid, to the parenchymal cells and in liver regeneration. As the group of cells that form the inner lining of the capillaries of the liver sinusoids, and being the first to be in contact with blood-borne particles, pathogens, and xenobiotics, they are prone to the deleterious effects of these. The aims of this thesis were to investigate the unique features of human liver sinusoidal endothelial cells (HLSEC) in comparison with endothelial cells from other vascular beds, evaluate the sensitivities of HLSEC to a range of hepatotoxic drugs, including small-molecule receptor tyrosine kinase inhibitors (RTKIs), such as regorafenib, and to explore the role of HLSEC in a triculture human liver microtissue. Results obtained from this study showed that HLSEC expressed phenotypic features of vascular and lymphatic endothelial cells, particularly vascular endothelial growth factor receptor 2 (VEGFR-2) which could be activated by VEGF-A to stimulate cell proliferation, migration and tubular morphogenesis. HLSEC also expressed functional VEGFR-3. Transcriptomic analysis indicated that HLSEC expressed specialised genes, such as plasmalemma vesicle associated protein (PLVAP), that support its liver-specific structure and functions. HLSEC were more sensitive to a range of small-molecule receptor tyrosine kinase inhibitors than other hepatic cells. (primary human hepatocytes [PHH] and human hepatic fibroblasts [HHF]) and endothelial cells from other vascular beds (human dermal microvascular endothelial cells and human dermal lymphatic endothelial cells). Regorafenib inhibited the activation of VEGFR-2 thereby abrogating cell proliferation, migration, tubular morphogenesis as well as upregulation of angiocrine factors involved in liver regeneration following activation by vascular endothelial growth factor A (VEGF-A). Regorafenib also caused a disruption of cytoskeletal structure of HLSEC and induced apoptosis via activation of caspase 3. Triculture liver microtissues formed with PHH, HLSEC and HHF were vascularised with higher expression of liver-specific drug-metabolising enzymes in comparison with the same combination of cells cultured as a monolayer. However, metabolic competence of triculture liver microtissues was significantly lower than in their monoculture counterparts (consisting of PHH only). This study has further confirmed the uniqueness of HLSEC as a specialised endothelial cell adapted to its anatomical role, which could respond to a range growth factors to initiate endothelial cell-specific functions. It has also been demonstrated that HLSEC are a direct target of hepatotoxic drugs. Triculture liver microtissues generated with PHH, HLSEC and HHF showed less metabolic competence than their PHH-only counterparts. Future studies need to investigate the role of RTKIs in vascular toxicity using in vivo models of sinusoidal obstruction syndrome (SOS) and liver regeneration. Finally, it would be informative to investigate the possibility of identifying HLSEC-specific biomarkers of liver toxicity.