Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study.

Liver Sinusoidal Endothelial Cells (LSEC) line the hepatic vasculature providing blood filtration via transmembrane nanopores called fenestrations. These structures are 50-300 nm in diameter, which is below the resolution limit of a conventional light microscopy. To date, there is no standardized method of fenestration image analysis. With this study, we provide and compare three different approaches: manual measurements, a semi-automatic (threshold-based) method, and an automatic method based on user-friendly open source machine learning software. Images were obtained using three super resolution techniques - atomic force microscopy (AFM), scanning electron microscopy (SEM), and structured illumination microscopy (SIM). Parameters describing fenestrations such as diameter, area, roundness, frequency, and porosity were measured. Finally, we studied the user bias by comparison of the data obtained by five different users applying provided analysis methods.

Functionality based method for simultaneous isolation of rodent hepatic sinusoidal cells.

Chronic liver disease is the result of long term exposure to viruses or toxins such as alcohol, fat and drugs, and forms the basis for the development of liver fibrosis and primary liver cancer. In vitro and in vivo models are key to study the pathways involved in chronic liver disease and for the development of therapeutics. 3D co-culture systems are becoming the in vitro standard, which requires freshly isolated primary hepatic cells. We developed a novel isolation method to simultaneously isolate liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs) and hepatic stellate cells (HSCs). The method exploits the scavenging activity of LSECs, the phagocytic capacity of KCs and the retinoid content of HSCs in vivo to enable direct processing by fluorescence-activated cell sorting without additional antibody binding and washing steps. UFACS3, for UV-FACS-based isolation of 3 non-parenchymal liver cell types, yields functional and pure LSECs (98 ± 1%), KCs (98 ± 1%) and HSCs (97 ± 3%), with less hands-on time from healthy and diseased rodent livers. This novel approach allows a fast and effective combined isolation of sinusoidal cells for further analysis.

High-affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats.

UNLABELLED: Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. ABSTRACT: Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.