Novel screening methods for nanoscale changes in liver cell fenestrations elicited by pharmaceuticals

Abstract

This thesis focuses on liver sinusoidal endothelial cells (LSEC) and one of their characteristic features called fenestrations. LSEC line the hepatic sinusoids and filter solutes from the plasma. The bidirectional transport of solutes between the bloodstream and the interior of the liver is facilitated via transcellular nanopores called fenestrations. Fenestrations are dynamic structures with diameters of 50-350 nm that can respond to various drugs and adapt their diameter and/or number within minutes or even seconds. Both the number and diameter of fenestrations are important for the maintenance of proper liver function. Various agents were shown to influence LSEC fenestrations and here we gathered the literature about recreational and prescription drugs, as well as other agents, and their effects on fenestrations. This review also provides different hypotheses about fenestration structure, regulation, and dynamics as well as analyses of previously used imaging techniques and image analysis methods. In the next study, the application of machine learning and batch processing enabled the analysis of large datasets of hundreds or even thousands of images. Those methods were used in the next studies to quantitatively describe LSEC morphology. The differences in the reported values of parameters describing fenestrations depend mainly on the used imaging technique but also on sample preparation and image analysis method. In the third study, the combination of different types of microscopies in a correlative manner allowed a better understanding of those differences. The fenestration size can be influenced by the different types of fixation, especially by the drying-related shrinkage of cell body which results in about 30% increase in fenestration diameter. In the final part, the influence of xanthines was studied with the application of the previously optimized imaging techniques and image analysis methods. Xanthines are found in food and beverages but also in asthma medications. The results showed that in physiologically relevant concentrations (8-20 µg/ml, achievable by consumption of xanthine-containing products) xanthines have no negative effects and theobromine increased the number of fenestrations. In high concentrations all xanthines increased fenestration number which may find future applications using targeted delivery to avoid systemic side effects.