Peter McCourt

Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues

MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mphi2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18-20 epidermal-growth-factor domains, one X-link domain and three to six B-(X(7))-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mphi2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mphi2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mphi2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell-cell and cell-matrix interactions in vascular function and inflammatory processes.

The scavenger endothelial cell: a new player in homeostasis and immunity

To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste. This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 μm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

The mannose receptor on murine liver sinusoidal endothelial cells is the main denatured collagen clearance receptor

The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180‐kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose‐receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild‐type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild‐type mice, and denatured collagen uptake in wild‐type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. Conclusion: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs. (HEPATOLOGY 2007.)

Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.

Fenestrations are pores in liver sinusoidal endothelial cells that filter substrates and debris between the blood and hepatocytes. Fenestrations have significant roles in aging and the regulation of lipoproteins. However their small size (<200 nm) has prohibited any functional analysis by light microscopy. We employed structured illumination light microscopy to observe fenestrations in isolated rat liver sinusoidal endothelial cells with great clarity and spatial resolution. With this method, the three-dimensional structure of fenestrations (diameter 123+/-24 nm) and sieve plates was elucidated and it was shown that fenestrations occur in areas of abrupt cytoplasmic thinning (165+/-54 nm vs. 292+/-103 nm in non-fenestrated regions, P<0.0001). Sieve plates were not preferentially co-localized with fluorescently labeled F-actin stress fibers and endothelial nitric oxide synthase but appeared to occur in primarily attenuated non-raft regions of the cell membrane. Labyrinthine structures were not seen and all fenestrations were short cylindrical pores. In conclusion, three-dimensional structured illumination microscopy has enabled the unlimited power of fluorescent immunostaining and co-localization to reveal new structural and functional information about fenestrations and sieve plates.

Role of liver sinusoidal endothelial cells and stabilins in elimination of oxidized low-density lipoproteins

Atherogenesis is associated with elevated levels of low-density lipoprotein (LDL) and its oxidized form (oxLDL) in the blood. The liver is an important scavenger organ for circulating oxLDLs. The present study aimed to examine endocytosis of mildly oxLDL (the major circulating form of oxLDLs) in liver sinusoidal endothelial cells (LSECs) and the involvement of the scavenger receptors stabilin-1 and stabilin-2 in this process. Freshly isolated LSECs, Kupffer cells (KCs), and stabilin-1- and stabilin-2-transfected human embryonic kidney cells were incubated with fluorescently labeled or radiolabeled oxLDLs [oxidized for 3 h (oxLDL(3)), 6 h, or 24 h (oxLDL(24))] to measure endocytosis. The intracellular localization of oxLDLs and stabilins in LSECs was examined by immunofluorescence and immunogold electron microscopy. Whereas oxLDL(24) was endocytosed both by LSECs and KCs, oxLDL(3) (mildly oxLDL) was taken up by LSECs only. The LSEC uptake of oxLDLs was significantly inhibited by the scavenger receptor ligand formaldehyde-treated serum albumin. Uptake of all modified LDLs was high in stabilin-1-transfected cells, whereas stabilin-2-transfected cells preferentially took up oxLDL(24), suggesting that stabilin-1 is a more important receptor for mildly oxLDLs than stabilin-2. Double immunogold labeling experiments in LSECs indicated interactions of stabilin-1 and stabilin-2 with oxLDL(3) on the cell surface, in coated pits, and endocytic vesicles. LSECs but not KCs endocytosed mildly oxLDL. Both stabilin-1 and stabilin-2 were involved in the LSEC endocytosis of oxLDLs, but experiments with stabilin-transfected cells pointed to stabilin-1 as the most important receptor for mildly oxLDL.

Liver sinusoidal endothelial cells depend on mannose receptor‐mediated recruitment of lysosomal enzymes for normal degradation capacity

Liver sinusoidal endothelial cells (LSECs) are largely responsible for the removal of circulating lysosomal enzymes (LE) via mannose receptor (MR)‐mediated endocytosis. We hypothesized that LSECs rely on this uptake to maintain their extraordinarily high degradation capacity for other endocytosed material. Circulatory half‐life studies of 125I‐cathepsin‐D in MR knockout (MR−/−) and wild‐type mice, and endocytosis studies in LSEC cultures, showed a total dependence on the MR for effective clearance of cathepsin‐D. Radioiodinated formaldehyde‐treated serum albumin, a ligand for the LSEC scavenger receptors, was used to study catabolism of endocytosed material in MR−/− and wild‐type mice. The plasma clearance, liver uptake, and the starting point for release of degradation products to blood, were similar in both experimental groups, indicating normal endocytosis and intracellular transport of scavenger receptor ligands in MR−/− mice. However, the rate of formaldehyde‐treated serum albumin catabolism in the liver of the MR deficient animals was reduced to approximately 50% of wild‐type values. A similar reduction in intracellular degradation was recorded in LSEC cultures from MR−/− mice compared to wild‐type controls. In accordance with this, MR−/− LSECs had markedly and significantly reduced enzyme activities for four out of five LE tested, i.e., cathepsin‐D, α‐mannosidase, β‐hexosaminidase and arylsulfatase, but not acid phosphatase, compared to wild‐type controls. Immunoblot analysis showed that the content of pro‐cathepsin‐D relative to total cathepsin‐D in wild‐type LSECs was less than one‐fifth of that in hepatocytes, indicating lower endogenous LE production in the LSECs. Conclusion: We show for the first time that LSEC depend on MR‐mediated recruitment of LE from their surroundings for effective catabolism of endocytosed macromolecules. (HEPATOLOGY 2008;48:2007–2015.)

Optical trapping and propulsion of red blood cells on waveguide surfaces

We have studied optical trapping and propulsion of red blood cells in the evanescent field of optical waveguides. Cell propulsion is found to be highly dependent on the biological medium and serum proteins the cells are submerged in. Waveguides made of tantalum pentoxide are shown to be efficient for cell propulsion. An optical propulsion velocity of up to 
6 µm/s on a waveguide with a width of ~6 µm is reported. Stable optical trapping and propulsion of cells during transverse flow is also reported.

How does the hyaluronan scrap-yard operate?

Hyaluronan is an extracellular polysaccharide found throughout the extracellular matrix, especially in soft connective tissue. It has an unusual feature, in that its turnover rate is much greater than that of other extracellular matrix components. The mechanisms of its synthesis at the plasma membrane (by hyaluronan synthases) and lysosomal degradation (by hyaluronidases) are well documented. However, the mechanisms by which it enters those cells primarily involved in its degradation remain a mystery. Recent work now suggests that a novel scavenger receptor expressed on the surface of liver endothelial cells is responsible for part of this degradative process. Further study is required to fill the remaining gaps in our knowledge about this process in other tissues.

Stabilins are expressed in bone marrow sinusoidal endothelial cells and mediate scavenging and cell adhesive functions.

Bone marrow sinusoidal endothelial cells have a specific function as a site of transmigration of hematopoietic stem and progenitor cells and mature blood cells between bone marrow and blood stream. However, the specific characteristics of bone marrow sinusoidal endothelial cells are still largely unclear. We here report that these cells express stabilin-1 and stabilin-2, which in liver sinusoidal endothelial cells have been identified as endocytic scavenger receptors for several ligands, including SPARC and hyaluronan. We show here that intravenously injected formaldehyde-treated serum albumin, advanced glycation end-products, and collagen I alpha-chains were taken up by bone marrow sinusoidal endothelial cells, showing that these cells have a scavenging function and thereby may modulate bone marrow vascular stem cell niches. Importantly, we show hyaluronan mediated adhesion of hematopoietic stem and progenitor cells to stabilin-2-transfected cells, suggesting that stabilin-2 contributes to adhesion and homing of circulating stem and progenitor cells to bone marrow.

Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations

Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations.