Svistounov D, Warren A, McNerney GP, Owen DM, Zencak D, Zykova SN, et al. The relationship between fenestrations, sieve plates and rafts in liver sinusoidal endothelial cells. PLoS One 2012;7:e46134. (Reprinted with permission.)
Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.
The endothelial cells are a specialized cell type that line blood and lymphatic vessels and form a monostratified layer called the endothelium. The endothelium may be continuous or discontinuous, and in some tissues the communication between the parenchyma and blood circulation can be finely tuned by the presence of special transcellular pores called fenestrations.1 Thanks to the pioneering work performed by Wisse et al.2, 3 on the ultrastructure of liver sinusoids, we know that the liver sinusoidal endothelial cells (LSECs) contain fenestrations with diameters of ∼20-250 nm and without diaphragms that are arranged in special structures called sieve plates.
Several studies have stressed the importance of these special structural features of the LSEC in pathological conditions. For example, liver fibrosis and cirrhosis are associated with molecular and morphological changes of LSEC. Preclinical studies have demonstrated that LSECs undergo defenestration as an early event that precedes liver fibrosis. This pathological change, collectively with the formation of a continuous lamina basal, is called capillarization and is thought to contribute to the increment of intrahepatic resistance, hepatocellular necrosis, and hepatic stellate cell activation.4, 5 Atherosclerosis is another clinical condition that has been associated with variability in the diameter and number of fenestrations existing in LSEC. The chylomicron-remnants, formed by the metabolism of dietary lipids, must pass through the LSEC to be metabolized by the liver parenchyma. However, only small chylomicrons (i.e., smaller than 250 nm in diameter) have access to the space of Disse, a phenomenon referred to as sieving.3 The experimental evidence supporting this association derives from studies performed in experimental models of nicotine dosage and partial hepatectomy in rats.6, 7 Other indirect evidence that seems to point in this direction is the association between fenestration variability and the susceptibility of species-dependent hypercholesterolemia after dietary manipulation. In this context, animals that more easily develop atherosclerosis and hyperlipoproteinemia are precisely those that exhibit fewer and smaller fenestrations, such as rabbits and chickens.1
Despite these clinical implications, the publications related to this field are not abundant, likely due to the technological complexity required to visualize fenestrations in LSEC. Due to the resolution limit of nonconfocal light microscopy (close to the maximum diameter of the LSEC fenestration), the only useful tool for exploring this area is electron microscopy. These microscopes are designed to have a high resolution at the expense of processing the tissues and cells through fixation techniques that may modify the association between fenestrations and other membrane structures. Therefore, due to these methodological limitations, the molecular and structural basis of fenestration formation remains unknown.
With the goal of going beyond some of these methodological limitations, Svistounov et al.8 recently reported a new method to overcome the resolution barriers of optical microscopy in the study of fenestrations. Using three-dimensional structured illumination fluorescence light microscopy (3D-SIM), they were able to see how fenestrations organize in a primary culture of mouse LSEC while simultaneously studying the distribution of the raft and nonraft membrane microdomains. 3D-SIM is a form of light microscopy that relies on the creation of interference patterns from the use of fluorescent probes and that allows the visualization of cellular structures below the diffraction limit. With this methodology, the authors demonstrated that there was an inverse association between membrane rafts and sieve plates in LSEC. The localization of membrane rafts was predominantly in the perinuclear region, whereas the localization of the sieve plates was mainly peripheral. In addition, the authors assessed the effects of membrane raft manipulation on fenestrations. Specifically, they were able to demonstrate that by increasing the membrane raft percentage in LSEC, by treating cells with low doses of Triton X-100, they were able to lower the number of fenestrations in the plasma membrane. Consistently, a reduction in the stability of the membrane rafts, either using 7-ketocholesterol or by treating cells with actin-disrupting drugs, such as cytochalasin D, increased the number of fenestrations. The enhanced formation of fenestrations induced by cytochalasin-D was blocked and reversed by Triton X-100 treatment. In view of these results, the authors propose a model, the sieve-raft theory, that explains the formation of fenestrations in LSECs. In brief, some areas of the plasma membrane, which are devoid of membrane stabilizers, such as rafts or actin, invaginate. However, due to the thinness of the cytoplasmatic extensions in LSEC, these invaginations give rise to fenestrations instead of other types of cell vesicle structures (Fig. 1). The mechanism of action of vascular endothelial growth factor (VEGF), which has previously been reported to be involved in the regulation of fenestrations,9 is also consistent with this theory. Svistounov et al.8 showed that VEGF treatment was associated with a significant increase in the abundance of nonraft lipid regions on the cell membrane, confirming the inverse relationship between raft and fenestration.
The clinical implications of this study may be relevant. On the one hand, these results may help clarify some of the mechanisms underlying diseases related to hypercholesterolemia. It is reasonable to speculate that under hypercholesterolemic conditions, the endothelial cells would have a higher number of rafts microdomains on their plasma membranes, resulting in a reduction in fenestrations. Therefore, the effects of hypercholesterolemia on LSEC cells could accelerate the development of abnormal levels of circulating lipids, characteristic of atherosclerosis. Although in vivo data in this study partially confirm the effects observed in vitro, experimental studies in models of lipid metabolism disorders would have been desirable to confer clinical relevance to the raft-fenestration crosstalk described by the authors. On the other hand, the loss of fenestration has also been reported in the context of alcohol liver disease. Several authors have demonstrated that ethanol exposure modifies cell membrane fluidity in both in vitro and in vivo models of alcohol exposure.10 Moreover, liver cirrhosis has been associated with increased caveolin-1 expression, a protein closely related to lipid rafts, in endothelial cells.11 Therefore, there are reasons to consider that the sieve-raft theory is also applicable in the context of liver diseases. However, to validate the sieve-raft theory in this pathological condition, the existence of lipid raft enrichment in the membrane of LSEC must be fully demonstrated in diseased liver.
Despite the absence of experiments in pathological experimental models, this study is a major advance in our understanding of the mechanisms that regulate the formation of sieve plates and fenestrations in LSEC. This knowledge, together with the use of 3D-SIM or a similar technology, may help boost the research in this field and build a foundation for future therapeutic strategies.