Med Mol Morphol (2008) 41:1–4 DOI 10.1007/s00795-007-0390-7

© The Japanese Society for Clinical Molecular Morphology 2008

AWARD REVIEW

Hiroaki Yokomori

New insights into the dynamics of sinusoidal endothelial fenestrae in liver sinusoidal endothelial cells

Received: October 30, 2007 / Accepted: November 5, 2007

Abstract Ultrastructural studies have shown that liver sinusoidal endothelial cells (LSECs) contain a cytoskeletal framework of filamentous actin, and that the presence of actin in the form of a calmodulin–actomyosin complex is responsible for regulation of the diameter of sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently of cell morphology. We investigated actin filaments in relation to SEF in LSEC using heavy meromyosin decorated reaction and elucidated the roles of Rho and actin cytoskeleton in morphological and functional alterations of SEF. Second, according to intracytoplasmic Ca2+ concentration, plasma membrane Ca2+Mg2+-ATPase activities were clearly demonstrated on the outer surface of the labyrinth-like SEF in the isolated LSECs. Furthermore, by investigating intracytoplasmic Ca2+ concentration, we have demonstrated plasma membrane Ca2+-Mg2+-ATPase activities on the outer surface of the labyrinth-like SEF in the isolated LSECs. Currently, the majority of fenestral studies are focused on finding ways to increase the liver sieve’s porosity, which is reduced through pathological mechanisms. Key words Rho · Caveolin · Actin · Sinusoidal endothelial cell · Sinusoidal endothelial fenestrae

Introduction The liver sinusoids can be regarded as unique capillaries that differ from other capillaries by the presence of open pores or fenestrae lacking a diaphragm and a basal lamina underneath the endothelium.1 The fenestral diameter decreases slightly from the periportal to centrilobular zone, whereas the number increases from the periportal to centrilobular zone.2 The diameter and number of fenestrae are H. Yokomori (*) Kitasato Institute Medical Center Hospital, 6-100 Arai, Kitamoto, Saitama 364-8501, Japan Tel. +81+485-93-1212; Fax +81+485-93-1239 e-mail: [email protected]

not static but change dynamically and are influenced by a variety of agents.3 Structural integrity of the sinusoidal endothelial fenestrae (SEF) is believed to be essential for the maintenance of normal exchanges of fluids, solutes, particles and metabolites between the parenchymal cells and the sinusoidal blood. Through impairment of substrate exchange, defenestration of the liver sinusoidal endothelial cell (LSEC) is a major contributor to hepatic dysfunction in liver cirrhosis4 and aging.5,6

Sinusoidal endothelial fenestral formation Caveolae are uncoated plasmalemmal invaginations 50– 100 nm in diameter, which exist in various cell types. Several functions have been ascribed to caveolae, including transcytosis in capillary endothelial cells, potocytosis of small molecules and ions, regulation of cytoplasmic Ca2+ concentration, and signal transduction.7 Intracytoplasmic free calcium ions (Ca2+) are maintained at a very low concentration in mammalian tissue by the extrusion of Ca2+ across a steep extracellular Ca2+ gradient, mainly through the activity of plasma membrane Ca2+ pump ATPase.8 Immunogold electron microscopy of cryosections revealed that locations of calcium pumps correspond to caveolae, or smooth invaginations of the plasma membrane.9 Serial ultrathin sectioning studies showed that almost all apparent free vesicles in capillary endothelial cells are actually caveolae with a limiting membrane that is continuous with the plasma membrane.10 This concept should be viewed with caution; stating that fenestrae correspond to racemose invaginations may somehow create confusion because there are also data that either clearly or indirectly indicate a possible relationship between caveolae, vesiculovacuolar organelles, and diaphragmed fenestrae.11 Furthermore, electron microscopic studies have also demonstrated caveolin-1 in caveolae and fused clustered vesicles, also referred to as vesiculovacuolar organelles (VVOs). VVOs are clusters of cytoplasmic vesicles and vacuoles that span endothelial cytoplasm from the lumen to the albumen, transendothelial

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Fig. 1. Cytochemical reaction products of Ca2+-Mg2+-ATPase activity are localized on the outer surface of the plasma membrane of the labyrinth-like sinusoidal endothelial fenestrae (SEF)15

cell pores, and the fenestrae.12 In addition, the plasma membrane-invaginated, labyrinth-like structures of the SEF interconnect with one another, and racemose clusters of caveolae have been identified in the vascular endothelial cell in a state of vascular endothelial growth factor (VEGF)induced increased permeability.12 By serial sections, the electron microscopic approach can perhaps show a functional or structural relationship between nondiaphragmed fenestrae formation and caveolae in LSECs, as has been demonstrated in the case of diaphragmed fenestrae. Moreover, we reported transmission electron microscopic findings showing that SEF were labyrinth-like structures, and that cytochemical reaction products of Ca2+-Mg2+ ATPase activity are located around the labyrinth-like structures of the SEF13–15 (Fig. 1).

Cytoskeleton of sinusoidal endothelial fenestrae Actin microfilaments and calmodulin are closely associated with the endothelial plasma membrane, suggesting that in the presence of calcium ions and calmodulin, actin microfilaments may be involved in the contraction and dilatation of endothelial cell fenestrae. Oda et al.16 described the presence of actin filaments in the neighborhood of fenestrae, and postulated that the cytoskeleton of LSEC plays an important role in the modulation of fenestrae. In the following years, this notion was supported by several authors; all confirmed the presence of actin,17 myosin,18 and calmodulin19 in LSECs. Moreover, in their whole-mount electron microscopic studies of LSEC, Braet et al.20 had added the following new insights regarding the structure and function of the cytoskeleton in SEF areas. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton ring (FACR). Using the actin inhibitors misakinolide and dihydrohalichondramide, Braet et al.21 demonstrated structures in LSECs indicative of fenestrae formation and designated

Fig. 2. Transmission electron micrograph of rat liver sinusoidal endothelial cell after heavy meromyosin decorated reaction. Actin filaments are identified by the characteristic “arrowhead decoration” of thin filaments aligning close to the SEF. Arrowheads, actin filaments. Bar 100 nm23

them fenestrae-forming center (FFC). Knowledge of the status of actin organization at specific cellular locations and at specific times is required to understand the process of fenestrae formation. The effects of the actin-disrupting marine toxin latrunculin A on the cytoskeleton and fenestrae of LSECs were investigated. Scanning electron microscopic preparations showed that latrunculin A almost doubles the number of fenestrae within 10 min.22 Antimycin A-induced defenestration is associated with the development of an elevated structure within the sieve plate. Reduction of fenestrae may occur via these golf ball-like structures that have been termed “defenestration center (DFC).”20 When LSECs were reacted with 0.1% Triton X and 15% glycerinated PHEM buffer containing heavy meromyosin decorated reaction, actin filaments were clearly demonstrated around SEF in LSEC23 (Fig. 2). Rho regulates the cytoskeleton by triggering the assembly of cytoplasmic stress fibers composed of filamentous actin and focal adhesions involving a complex containing vinculin.24 Treatment of LSECs with the Rho promoter lysophosphatidic acid (LPA) contracts the SEF, concomitant with increases in Factin stress fiber and actin microfilament and high expression of phosphorylated myosin light chain kinase. Following treatment with Clostridium botulinum C3-like transferase (an inhibitor of Rho protein), SEF dilate and fuse, concomitant with a loss of F-actin and microfilament and low expression of phosphorylated myosin light chain. These results indicate that Rho modulates fenestral changes in SEC via regulation of the actin cytoskeleton.25 In a study on the role of actin filaments in cell contraction of hepatocyte couplets using cytoskeleton-enriched

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canalicular membranes, Tsukada at al.26 showed that the Ca2+/ATP-induced contraction of microfilaments associated with bile canalicular membranes is a dynamic process resulting in narrowing or complete closure of the canalicular lumen. Therefore, it is important to investigate cytoskeleton-rich membrane when studying the contraction of SEF in LSECs.

Clinical aspects Aging is accompanied by a change in LSEC phenotype, with loss of fenestration but less extensive changes in the basement membrane. Given the limited changes in the basement membrane, the change in SEC phenotype has been referred to as pseudocapillarization.5,6,27 Deleve et al.28 demonstrated the appearance of CD31 surface expression on hepatic sinusoidal liver endothelial cells that had lost fenestration after 3 days in culture. CD31 becomes detectable in the process of cirrhosis, and the expression of this adhesion molecule correlates inversely to fenestration pattern. CD31 localization may be used as a marker of SEC phenotype to examine the paracrine and autocrine regulation of the SEC phenotype. The data gathered so far have already alerted scientists to the possibility of changes of SEF in physiological and pathological processes such as aging and cirrhosis. Recently, liver organoid has been reconstructed in a radial-flow bioreactor by coculturing mouse immortalized sinusoidal endothelial and hepatic stellate cell lines. Study using liver organoid suggests that endothelial gap junctional intercellular communication may regulate fenestral number in immortalized endothelial cells.29 Furthermore, communication between sinusoidal endothelial cells and hepatic stellate cells is also an important issue and should be further examined. Dr. Hiroaki Yokomori, of the Department of Internal Medicine, Kitasato Medical Center Hospital, Saitama, Japan, is the winner of the Japanese Society for Clinical Molecular Morphology Award for Promoting Young Researchers in 2007. Dr. Yokomori was recognized for his great contribution in elucidating the role of sinusoidal endothelial fenestrae in the physiology and pathology of the liver.

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