Phenotypic changes in rat liver sinusoidal endothelial cells during early stages of in vitro culture

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells with a unique fenestrated morphology and important functions in blood waste clearance and liver innate immunity. A striking characteristic of... [ view full abstract ]

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells with a unique fenestrated morphology and important functions in blood waste clearance and liver innate immunity. A striking characteristic of the cells is their very high endocytic capacity compared to other endothelial cells. LSEC morphology and function are affected by ageing and liver disease. However, a major challenge when studying these cells ex vivo is their rapid dedifferentiation in traditional culture systems with loss of cell fenestrations and decreased endocytosis. The aim of the present study was to reveal LSEC pathways that are affected the first 24h in in vitro adherent cultures, and study the effect of dexamethasone on cell survival, phenotype, and function. Purified LSECs, freshly isolated by collagenase perfusion of rat liver, were cultured under hypoxic conditions in a DMEM-based medium with serum-free supplements, including antioxidants. Comparative expression of cell-associated proteins was done by MS/MS using tandem mass tag (TMT) technology. LSEC fenestration was assessed by scanning electron microscopy, and cell function by endocytosis of ¹²⁵I- or fluorescently labeled ligands. Proteomic analyses of LSEC cultures at 2h and 24h showed development towards an inflammatory phenotype, with upregulation of CxCR4, and ICAM-1, and loss of LSEC-markers such as Lyve1. The 24h cultures showed preserved endocytosis, but decreased rate of intracellular degradation of the test ligand, compared to 2h cultures. Dexamethasone highly improved LSEC survival after 24h (number of adherent cells), and also had positive effects on the cell morphology.