No other multimodal biomedical high-resolution imaging technique to-date has allowed collecting new fine structural information on the lifespan, formation, and disappearance of LSEC fenestrae. By doing so, we gathered also evidence of three different pathways implemented in the loss of fenestrae that result in defenestrated LSECs. This article is protected by copyright. All rights reserved.

Here, we report an atomic force microscopy (AFM)-based imaging method for resolving the fine nanostructures (e.g., fenestrations) in the membranes of live primary murine liver sinusoidal endothelial cells (LSECs). From data on topographical and nanomechanical properties of the selected cell areas collected within 1 min, we traced the dynamic rearrangement of the cell actin cytoskeleton connected with the formation or closing of cell fenestrations, both in non-stimulated LSECs as well as in response to cytochalasin B and antimycin A. In conclusion, AFM-based imaging permitted the near real-time measurements of dynamic changes in fenestrations in live LSECs.

Healthy liver sinusoidal endothelial cells (LSECs) maintain liver homeostasis, while LSEC dysfunction was suggested to coincide with defenestration. Here, we have revisited the relationship between LSEC pro-inflammatory response, defenestration, and impairment of LSEC bioenergetics in non-alcoholic fatty liver disease (NAFLD) in mice. We characterized inflammatory response, morphology as well as bioenergetics of LSECs in early and late phases of high fat diet (HFD)-induced NAFLD. LSEC phenotype was evaluated at early (2–8 week) and late (15–20 week) stages of NAFLD progression induced by HFD in male C57Bl/6 mice. NAFLD progression was monitored by insulin resistance, liver steatosis and obesity. LSEC phenotype was determined in isolated, primary LSECs by immunocytochemistry, mRNA gene expression (qRT-PCR), secreted prostanoids (LC/MS/MS) and bioenergetics (Seahorse FX Analyzer). LSEC morphology was examined using SEM and AFM techniques. Early phase of NAFLD, characterized by significant liver steatosis and prominent insulin resistance, was related with LSEC pro-inflammatory phenotype as evidenced by elevated ICAM-1, E-selectin and PECAM-1 expression. Transiently impaired mitochondrial phosphorylation in LSECs was compensated by increased glycolysis. Late stage of NAFLD was featured by prominent activation of pro-inflammatory LSEC phenotype (ICAM-1, E-selectin, PECAM-1 expression, increased COX-2, IL-6, and NOX-2 mRNA expression), activation of pro-inflammatory prostaglandins release (PGE 2 and PGF 2α ) and preserved LSEC bioenergetics. Neither in the early nor in the late phase of NAFLD, were LSEC fenestrae compromised. In the early and late phases of NAFLD, despite metabolic and pro-inflammatory burden linked to HFD, LSEC fenestrae and bioenergetics are functionally preserved. These results suggest prominent adaptive capacity of LSECs that might mitigate NAFLD progression.

The porosity of liver sinusoidal endothelial cells (LSEC) ensures bidirectional passive transport of lipoproteins, drugs and solutes between the liver capillaries and the liver parenchyma. This porosity is realized via fenestrations – transcellular pores with diameters in the range of 50–300 nm – typically grouped together in sieve plates. Aging and several liver disorders severely reduce LSEC porosity, decreasing their filtration properties. Over the years, a variety of drugs, stimulants, and toxins have been investigated in the context of altered diameter or frequency of fenestrations. In fact, any change in the porosity, connected with the change in number and/or size of fenestrations is reflected in the overall liver-vascular system crosstalk. Recently, several commonly used medicines have been proposed to have a beneficial effect on LSEC re-fenestration in aging. These findings may be important for the aging populations of the world. In this review we collate the literature on medicines, recreational drugs, hormones and laboratory tools (including toxins) where the effect LSEC morphology was quantitatively analyzed. Moreover, different experimental models of liver pathology are discussed in the context of fenestrations. The second part of this review covers the cellular mechanisms of action to enable physicians and researchers to predict the effect of newly developed drugs on LSEC porosity. To achieve this, we discuss four existing hypotheses of regulation of fenestrations. Finally, we provide a summary of the cellular mechanisms which are demonstrated to tune the porosity of LSEC.