Unraveling nanoscale alterations in liver cell fenestrations - Morphological studies via optical super-resolution microscopy approaches

Abstract

The endothelium makes up the innermost cell layer of blood vessels. It consists of a thin layer of simple squamous cells, forming an interface between circulating blood and the surrounding tissue. Endothelial cells of different vascular beds are specialized according to tissue-specific functions. For this project emphasis was placed upon high-resolution methods enabling the study of liver sinusoidal endothelial cells (LSECs) below the diffraction limit of visible light (~200 nm). LSECs have unusual morphology with as much of 20% of their surface covered with cellular fenestrations - holes through the cells of 50-300 nm diameter. These allow bi-directional flow of plasma from the sinusoids to the surrounding hepatocytes, while retaining blood cells in the sinusoidal lumen. Little is known about the function of fenestrations, their regulation, and their role in the transfer of metabolites, viruses, lipoproteins and pharmaceuticals to other cells of the liver. There are two major challenges with the study of LSEC fenestrations; i) the majority have diameters smaller than the diffraction limit of visible light and; ii) they disappear rapidly in cultured LSEC, and there are no cell line alternatives that express fenestrations. To address the first challenge, the project used classical super resolution imaging technologies such as scanning electron microscopy, and two novel super-resolution optical microscopy modalities: dSTORM (direct stochastic optical reconstruction microscopy) and SIM (structured illumination microscopy) to study the in vitro effects of xanthines, sildenafil and oxidized LDL on LSEC fenestrations. One of the xanthines, theobromine, and sildenafil increased both the frequency and diameter of fenestrations in cultured LSEC. While oxidized LDL caused major disruptions in LSEC fenestration morphology. Finally, to address the second challenge, namely the rapid loss of fenestrations in LSEC, a cryopreservation method for freshly isolated LSEC was developed such that they can be used at researchers’ convenience, rather than directly after isolation from live