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.

Oxidized albumin (oxHSA) is elevated in several pathological conditions, such as decompensated cirrhosis, acute on chronic liver failure and liver mediated renal failure. Patient derived oxidized albumin was previously shown to be an inflammatory mediator, and in normal serum levels of oxHSA are low. The removal from circulation of oxidized albumins is therefore likely required for maintenance of homeostasis. Liver sinusoidal endothelial cells (LSEC) are prominent scavenger cells specialized in removal of macromolecular waste. Given that oxidized albumin is mainly cleared by the liver, we hypothesized the LSEC are the site of uptake in the liver. In vivo oxHSA was cleared rapidly by the liver and distributed to mainly the LSEC. In in vitro studies LSEC endocytosed oxHSA much more than other cell populations isolated from the liver. Furthermore, it was shown that the uptake was mediated by the stabilins, by affinity chromatography-mass spectrometry, inhibiting uptake in LSEC with other stabilin ligands and showing uptake in HEK cells overexpressing stabilin-1 or -2. oxHSA also inhibited the uptake of other stabilin ligands, and a 2-h challenge with 100 µg/mL oxHSA reduced LSEC endocytosis by 60% up to 12 h after. Thus the LSEC and their stabilins mediate clearance of highly oxidized albumin, and oxidized albumin can downregulate their endocytic capacity in turn.

Background: Reactive oxygen species (ROS) are prevalent in the liver during intoxication, infection, inflammation, and aging. Changes in liver sinusoidal endothelial cells (LSEC) are associated with various liver diseases. Methods: Isolated rat LSEC were studied under oxidative stress induced by H ₂ O ₂ at different concentrations (0.5–1000 µM) and exposure times (10–120 min). LSEC functions were tested in a dose-dependent and time-dependent manner. Results: (1) Cell viability, reducing potential, and scavenging function decreased as H ₂ O ₂ concentration and exposure time increased; (2) intracellular ROS levels rose with higher H ₂ O ₂ concentrations; (3) fenestrations exhibited a dynamic response, initially closing but partially reopening at H ₂ O ₂ concentrations above 100 µM after about 1 hour; (4) scavenging function was affected after just 10 minutes of exposure, with the impact being irreversible and primarily affecting degradation rather than receptor-mediated uptake; (5) the tubulin network was disrupted in high H ₂ O ₂ concentration while the actin cytoskeleton appears to remain largely intact. Finally, we found that reducing agents and thiol donors such as n -acetyl cysteine and glutathione (GSH) could protect cells from ROS-induced damage but could not reverse existing damage as pretreatment with n -acetyl cysteine, but not GSH, reduced the negative effects of ROS exposure. Conclusions: The results suggest that LSEC does not store an excess amount of GSH but rather can readily produce it in the occurrence of oxidative stress conditions. Moreover, the observed thresholds in dose-dependent and time-dependent changes, as well as the treatments with n -acetyl cysteine/GSH, confirm the existence of a ROS-depleting system in LSEC.

Hepatic sinusoids are lined with thin endothelial cells with transcellular pores, termed fenestrations. These fenestrations are open channels that connect the sinusoidal lumen to the underlying Space of Disse (SoD) and the hepatocytes of the liver parenchyma. Fenestrations range from 0.05 to 0.35 µm in diameter and cover 5–15% of the sinusoidal endothelial surface area, depending on their location along the sinusoids. The direct measurement of hemodynamic parameters, such as pressure and flow velocity, remains challenging within the narrow sinusoids. Such knowledge would increase our understanding of the physiology of the hepatic niche and possible implications in aging or diseases in which fenestrations are reduced or lost. Few simulations of liver blood flow focus on the level of the individual sinusoid, and fewer still include the transcellular pores (fenestrations) of the sinusoidal endothelium. Furthermore, none have included (i) a porosity gradient along the sinusoid wall, modeled using through-all pores rather than a porous medium, (ii) the presence of the SoD, or (iii) lymphatic drainage. Herein, computed fluid dynamics (CFD) simulations were performed using a numerical model with relevant anatomical characteristics (length, diameter, porosity, inlet/outlet pressure, and lymphatic outflow from the portal region of the SoD). The greatest contribution to luminal velocity magnitude and pressure was the overall shape of the vessel. Divergent-radius models yielded velocity magnitudes 1.5–2 times higher than constant-radius models, and pressures were 5–8% lower in the divergent-radius models compared to the constant-radius models. Porosity only modestly contributed to luminal pressure. The luminal velocity magnitude was largely unaffected by the presence or absence of lymphatic drainage. Velocity magnitudes through fenestrations were lower in higher-porosity models (20%) vs. lower-porosity models (5%) across all models (0.4–0.55-fold lower). Velocity magnitudes through the space of Disse were increased 3–4 times via the addition of lymphatic drainage to the models, while pressures were decreased by 6–12%. The flow velocity in the SoD was modified via differences in porosity, while the flow velocity in the lumens of the sinusoids was largely unaffected. The overall shape of the vessel is the single most important factor in the pressure flow behavior of the sinusoidal lumen. The flow rate over hepatocytes and the SoD is modestly affected by the distribution of porosity along the sinusoid and greatly affected by the lymphatic drainage, parameters that would be of interest for modeling the exchange of blood with the hepatic parenchyma.