NEW OBSERVATIONS ON CYTOSKELETON AND FENESTRAE IN ISOLATED RAT-LIVER SINUSOIDAL ENDOTHELIAL-CELLS

Fenestrae control the exchange of fluids, dissolved compounds and smal l particles between the blood and the space of Disse, and are primaril y limited at one side by parenchymal cells. We recently described a si mple and rapid method for the isolation, purification and cultivation of rat liver sinusoidal endothelial cells. With regard to the purity a nd morphology of liver endothelial cells, a detailed microscopic study was performed. Purity and viability after selective adherence was 74 and 95%, respectively. Liver endothelial cell purity was further enhan ced to about 95% during adherence and spreading on collagen after 8 h of culture. Liver endothelial cells isolated by this method provide a viable cell population, enabling the study of structure and function o f these cells in vitro. We investigated the cytoskeleton associated wi th fenestrae and sieve plates of liver endothelial cells. Cultured cel ls were slightly fixed and treated with cytoskeleton extraction buffer containing 0.1% Triton. Whole mounts of extracted liver endothelial c ells were prepared for scanning and transmission electron microscopy. Extracted liver endothelial cells show an integral, intricate cytoskel eton. Sieve plates and fenestrae are clearly delineated by cytoskeleto n elements. Fenestrae are surrounded by a filamentous, fenestrae-assoc iated cytoskeleton ring with an average filament thickness of 16 nm. A dditionally, sieve plates are surrounded and delineated by microtubuli , which form a network together with additional branching cytoskeletal elements. Microtubuli are sometimes found delineating linear arrangem ents of fenestrae. In conclusion, liver endothelial cells possess a cy toskeleton, that defines and supports sieve plates and fenestrae. Fene strae-associated cytoskeleton rings are involved in determining the si ze of fenestrae. The fenestrae-associated cytoskeleton therefore proba bly controls the important hepatic function of endothelial filtration.