No abstract

This review provides a detailed overview of the current state of knowledge about the ultrastructure and dynamics of liver sinusoidal endothelial fenestrae. Various aspects of liver sinusoidal endothelial fenestrae regarding their structure, origin, species specificity, dynamics and formation will be explored. In addition, the role of liver sinusoidal endothelial fenestrae in relation to lipoprotein metabolism, fibrosis and cancer will be approached.

In normal rat liver, Kupffer cells were unequivocally identified using peroxidase cytochemistry by light microscopy in semithin plastic sections. The Kupffer cell population was found to constitute 31% of the sinusoidal cells and by morphometry and serial sectioning, a mean absolute number of 14 to 20 X 10(6) Kupffer cells per g liver was calculated. The mean distribution of Kupffer cells in the liver lobules was 43% in the periportal, 28% in the midzonal and 29% in the central area of the lobule. Administration of latex particles labeled only 64% of all Kupffer cells, and in particular centrally located cells, showed a lower activity of latex uptake, even at overloading doses. Furthermore, the latter cells were of smaller size than periportal Kupffer cell profiles. The mean number and distribution of latex-labeled Kupffer cells did not change over a period of 3 months, indicating a long lifetime for these resident macrophages. This slow population turnover was supported by the observed small mitotic index, 0.06% after a 6 hr arrest by vinblastine, and by the small [3H]thymidine labeling index which did not change over a period of 3 weeks after administration of the label. It is proposed that the Kupffer cell population, under physiologic conditions, is a long-living and self-renewing population, the kinetics of which substantially differ from those of other sinusoidal cell types.

SummaryCritical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.

Fenestrae allow the passage of gene transfer vectors from the sinusoidal lumen to the surface of hepatocytes. We have previously shown that the diameter of fenestrae correlates with species and strain differences of transgene expression following intravenous adenoviral transfer. In the current study, we demonstrate that the diameter of fenestrae in humans without liver pathology is 107 ± 1.5 nm. This is similar to the previously reported diameter in New Zealand White (NZW) rabbits (103 ± 1.3 nm) and is significantly smaller than in C57BL/6 mice (141±5.4 nm) and SpragueDawley rats (161 ± 2.7 nm). We show that the diameter of fenestrae in one male NZW rabbit and its offspring characterized by a more than 50-fold increase of transgene expression after adenoviral gene transfer is significantly (113 ± 1.5 nm; Po0.001) larger than in control NZW rabbits. In vitro filtration experiments using polycarbonate filters with increasing pore sizes demonstrate that a relatively small increment of the diameter of pores potently enhances passage of adenoviral vectors, consistent with in vivo data. In conclusion, the small diameter of fenestrae in humans is likely to be a major obstacle for hepatocyte transduction by adenoviral vectors.