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.     

Distribution of endothelin receptor subtypes ETA and ETB in the rat kidney.

The endothelin (ET) receptor system is markedly involved in the regulation of renal function under both physiological and pathophysiological conditions. The present study determined the detailed cellular localization of both ET receptor subtypes, ET(A) and ET(B), in the vascular and tubular system of the rat kidney by immunofluorescence microscopy. In the vascular system we observed both ET(A) and ET(B) receptors in the media of interlobular arteries and afferent and efferent arterioles. In interlobar and arcuate arteries, only ET(A) receptors were present on vascular smooth muscle cells. ET(B) receptor immunoreactivity was sparse on endothelial cells of renal arteries, whereas there was strong labeling of peritubular and glomerular capillaries as well as vasa recta endothelium. ET(A) receptors were evident on glomerular mesangial cells and pericytes of descending vasa recta bundles. In the renal tubular system, ET(B) receptors were located in epithelial cells of proximal tubules and inner medullary collecting ducts, whereas ET(A) receptors were found in distal tubules and cortical collecting ducts. Distribution of ET(A) and ET(B) receptors in the vascular and tubular system of the rat kidney reported in the present study supports the concept that both ET receptor subtypes cooperate in mediating renal cortical vasoconstriction but exert differential and partially antagonistic effects on renal medullary function.     

The three-dimensional cytoarchitecture of the interstitial tissue in the rat kidney

Summary The cytoarchitecture of the interstitial tissue of the rat kidney was studied by combined scanning and transmission electron microscopy. The renal interstitium is composed of an elaborate network of stellate sustentacular cells. In the cortex, sustentacular cells radiate thin branching processes to form a fine reticulum, which supports intertubular spaces. In the medulla, these cells extend thick processes horizontally along the basal surfaces of the thin limbs or vasa recta, reinforcing their attenuate walls. The horizontal processes connect with each other at their terminals, compartmentalizing the interstitial space into thin layers. The medullary sustentacular cells contain abundant small lipid droplets. The network of sustentacular cells houses vasa recta, keeping them in parallel position to each other and to the tubules. The arterial vasa recta are accompanied by pericytes, which frequently contain lipid droplets larger in size than those in the sustentacular cells. Venous vasa recta extend numerous basal microvilli, which anchor the venous wall to adjacent tubules or vessels. Numerous free cells, round in shape, are found in the sustentacular cell network, especially in the cortex. They consist of macrophages and occasional lymphocytes. Some macrophages extend long pseudopodia, while others make intimate contact with lymphocytes, suggesting their high level of activity.     

An immortalized human liver endothelial sinusoidal cell line for the study of the pathobiology of the liver endothelium

Background

The endothelium lines blood and lymph vessels and protects underlying tissues against external agents such as viruses, bacteria and parasites. Yet, microbes and particularly viruses have developed sophisticated ways to bypass the endothelium in order to gain access to inner organs. De novo infection of the liver parenchyma by many viruses and notably hepatitis viruses, is thought to occur through recruitment of virions on the sinusoidal endothelial surface and subsequent transfer to the epithelium. Furthermore, the liver endothelium undergoes profound changes with age and in inflammation or infection. However, primary human liver sinusoidal endothelial cells (LSECs) are difficult to obtain due to scarcity of liver resections. Relevant derived cell lines are needed in order to analyze in a standardized fashion the transfer of pathogens across the liver endothelium. By lentiviral transduction with hTERT only, we have immortalized human LSECs isolated from a hereditary hemorrhagic telangiectasia (HHT) patient and established the non-transformed cell line TRP3. TRP3 express mesenchymal, endothelial and liver sinusoidal markers. Functional assessment of TRP3 cells demonstrated a high capacity of endocytosis, tube formation and reactivity to immune stimulation. However, TRP3 displayed few fenestrae and expressed C-type lectins intracellularly. All these findings were confirmed in the original primary LSECs from which TRP3 were derived suggesting that these features were already present in the liver donor. We consider TRP3 as a model to investigate the functionality of the liver endothelium in hepatic inflammation in infection.     

An Intact Kidney Slice Model to Investigate Vasa Recta Properties and Function in situ

Background: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in 'live' kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. Results: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10-30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E(2)) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.     

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.     

Nuclear entry mechanism of the human polyomavirus JC virus-like particle: role of importins and the nuclear pore complex

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. Although transport of virions to the nucleus is an important step in JCV infection, the mechanism of this process has remained unclear. The outer shell of the JCV virion comprises the major capsid protein VP1, which possesses a putative nuclear localization signal (NLS), and virus-like particles (VLPs) consisting of recombinant VP1 exhibit a virion-like structure and physiological functions (cellular attachment and intracytoplasmic trafficking) similar to those of JCV virions. We have now investigated the mechanism of nuclear transport of JCV with the use of VLPs. Wild-type VLPs (wtVLPs) entered the nucleus of most HeLa or SVG cells. The virion structure of VLPs was preserved during transport to the nucleus as revealed by confocal microscopy of cells inoculated with fluorescein isothiocyanate-labeled wtVLPs containing packaged Cy3. The nuclear transport of wtVLPs in digitonin-permeabilized cells was dependent on the addition of importins alpha and beta and was prevented by wheat germ agglutinin or by antibodies to the nuclear pore complex. The nuclear entry of VLPs composed of VP1 with a mutated NLS was greatly inhibited, compared with that of wtVLPs, in both intact and permeabilized cells. Unlike wtVLPs, the mutant VLPs did not bind to importins alpha or beta. Limited proteolysis analysis revealed that the NLS of VP1 was exposed on the surface of wtVLPs. These results suggest that JCV VLPs bind to cellular importins via the NLS of VP1 and are transported into the nucleus through the nuclear pore complex.     

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.     

Liver sinusoidal endothelial cells are the principal site for elimination of unfractionated heparin from the circulation

The mechanism of elimination of blood borne heparin was studied. To this end unfractionated heparin (UFH) was tagged with FITC, which served as both a visual marker and a site of labeling with (125)I-iodine. UFH labeled in this manner did not alter the anticoagulant activity or binding specificity of the glycosaminoglycan. Labeled heparin administered intravenously to rats (0.1 IU/kg) had a circulatory t(1/2) of 1.7 min, which was increased to 16 min upon coinjection with unlabeled UFH (100 IU/kg). At 15 min after injection, 71% of recovered radioactivity was found in liver. Liver cell separation revealed the following relative uptake capacity, expressed per cell: liver sinusoidal endothelial cell (LSEC)-parenchymal cell-Kupffer cell = 15:3.6:1. Fluorescence microscopy on liver sections showed accumulation of FITC-UFH only in cells lining the liver sinusoids. No fluorescence was detected in parenchymal cells or endothelial cells lining the central vein. Fluorescence microscopy of cultured LSECs following binding of FITC-UFH at 4 degrees C and chasing at 37 degrees C, showed accumulation of the probe in vesicles located at the periphery of the cells after 10 min, with transfer to large, evenly stained vesicles in the perinuclear region after 2 h. Immunogold electron microscopy of LSECs to probe the presence of FITC following injection of FITC-UFH along with BSA-gold to mark lysosomes demonstrated colocalization of the probe with the gold particles in the lysosomal compartment. Receptor-ligand competition experiments in primary cultures of LSECs indicated the presence of a specific heparin receptor, functionally distinct from the hyaluronan/scavenger receptor (Stabilin2). The results suggest a major role for LSECs in heparin elimination.     

Liver Sinusoidal Endothelial Cells Escape Senescence by Loss of p19ARF

Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19^ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19^ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19^ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19^ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19^ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19^ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.     

Autoregulation of blood flow in renal medulla of the rat: no role for angiotensin II

Autoregulation of blood flow was assessed by a dual-slit technique in descending and ascending vasa recta of the exposed renal papillae of antidiuretic rats. There was complete autoregulation of blood flow in descending vasa recta. The lower limit of autoregulation was approximately 85 mmHg (1 mmHg = 133.3 Pa) and the upper limit was greater then 160 mmHg. Autoregulation in ascending vasa recta was also good. To test the role of angiotensin II in this autoregulation, the converting enzyme inhibitor captopril was infused. Captopril had no effect on autoregulation of blood flow in either descending or ascending vasa recta. We conclude that blood flow in vasa recta of renal medulla is efficiently autoregulated and that this autoregulation is independent of angiotensin II.     

Clathrin-coated vesicles form a unique net-like structure in liver sinusoidal endothelial cells by assembling along undisrupted microtubules

Liver sinusoidal endothelial cells (LSECs) are highly active professional scavenger cells using clathrin-mediated endocytosis to clear the blood from macromolecular waste products. Using confocal microscopy, we observed a remarkable net-like distribution of clathrin heavy chain (CHC) in LSECs while all other cell types examined including various primary endothelial cells and cell lines showed the well-known punctuate staining pattern representing clathrin-coated vesicles (CCV). The net-like distribution of CHC in LSECs co-localized fully with microtubules, but not with actin. Upon 3D imaging, the net-like distribution of CHC resolved into numerous CCVs organized along the microtubules. The CCVs only partially co-localized with early endosome antigen 1 (EEA1) and adaptor protein 2 (AP-2). Endocytic vesicles containing ligand destined for degradation (FITC-AHGG) were organized along the clathrin/tubulin net-like structures, whereas transferrin-containing recycling vesicles co-localized to a much lower extent. Disruption of the microtubules by nocodazole treatment caused a collapse of the net-like organization of CCVs as well as a profound redistribution of EEA1, AP-2 and FITC-AHGG-containing vesicles, while transferrin internalization and recycling remained unaffected.     

Long-term preservation of high endocytic activity in primary cultures of pig liver sinusoidal endothelial cells

Together with Kupffer cells, liver sinusoidal endothelial cells (LSECs) constitute the most powerful scavenger system in the body. However, studies on LSEC function are hampered by the fact that the cells lose their scavenger ability and start deteriorating after a few days in culture. The purpose of the present study was to improve the conditions of cultivation to prolong the survival of pig LSECs in vitro. We used the high capacity receptor-mediated endocytosis of soluble waste molecules as a marker for functionally intact cells in the cultures. Compared with two commercially-, and two other media specifically designed for use with either SECs or hepatocytes from rat, our newly developed serum-free medium, DM 110/SS, devoid of any components of animal origin, was superior in maintaining the endocytic activity. Of six growth factors studied for their effect on endocytosis, basic fibroblast, and recombinant epidermal, but not vascular endothelial growth factor, were found to be most beneficial. After 8 days in DM 110/SS, LSECs maintained endocytosis via the scavenger receptor, mannose receptor, collagen alpha-chain receptor and the Fc-gamma receptor. All endocytosed ligands, except for aggregated IgG were degraded in 8-day-old cultures. Using the new medium, the cells endocytosed ligands for up to 20 days, and survived for at least an additional 10 days, albeit without the high endocytic activity typical of intact LSECs. Importantly, DNA synthesis in prolonged cultures of LSECs was observed only when maintained in DM 110/SS medium. In conclusion, we describe a protocol for the maintenance of LSECs in culture for the longest period yet reported.     

Ultrastructural observations on the renal papilla of the rabbit

Summary Ultrastructural studies of renal papillae of New Zealand brown rabbits under different states of water balance indicate no morphological variation between control, antidiuretic and diuretic animals; the only exception being a decrease in the amount of glycogen in the collecting duct cells in the antidiuretic state and an increase in the diuretic.The light cells of the collecting ducts have a low electron density and show a paucity of organelles. These comprise mitochondria, Golgi apparatus, multivesicular bodies, sparse endoplasmic reticulum and free ribosomes. The centrally-placed, spherical nucleus demonstrates large numbers of nuclear pores. The lateral surfaces and bases of the cells have considerable infoldings which may have functional significance.The attenuated endothelial cells of the vasa recta are punctuated by fenestrations which are most frequently crossed by membrane. The cells contain micropinocytotic and pinocytotic vesicles.The loops of Henle in the papilla are lined by squamous cells which are extended longitudinally in the form of interdigitating processes. The bases of the cells of most loops are scalloped.The interstitial cells are embedded in an amorphous matrix containing occasional collagen fibres and strands of fibrillar material. The cells are irregular in outline and have moderately developed endoplasmic reticulum and Golgi apparatuses.Tight junctions between the cells of all collecting ducts, loops of Henle and vasa recta are a constant finding. All these tubular elements are surrounded by a prominent basement membrane; that associated with the loops of Henle tends to be multiplied, particularly at scalloped regions. The membrane associated with the vasa recta is single except at regions where it projects across the interstitium to the membranes of the collecting ducts and loops of Henle.The functional implications of these findings are discussed.     

Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.     

Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

Mammalian Genome - Stabilin2 (Stab2) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor...     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

Isolation and characterization of monopinocytotic vesicles containing polyomavirus from the cytoplasm of infected mouse kidney cells.

Monopinocytotic vesicles containing polyomavirus were isolated from the cytoplasm of mouse kidney cells infected with polyomavirus using sucrose density gradients. Nonenclosed, membrane-associated virions released by the action of neuraminidase separated from vesicle-enclosed virions in the sucrose gradient. Marker enzyme assays indicated the derivation of the vesicle membrane from the plasma membrane of the cell. The 125I-labeled virus enclosed in the vesicle sedimented more slowly in the gradient and was not observed unless infection and endocytosis had occurred. Detergent treatment of virion-containing vesicles caused the release of polyomavirus with sedimentation properties similar to those of purified polyoma virions. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virion proteins from vesicles containing virions demonstrated patterns of proteins similar to those of purified intact virions. Electron microscopy confirmed the presence of single intact virions inside vesicles. The study of these monopinocytotic virion-containing vesicles represents a further step in elucidating the early events of polyomavirus infection.     

Production of Factor VIII by Human Liver Sinusoidal Endothelial Cells Transplanted in Immunodeficient uPA Mice

Liver sinusoidal endothelial cells (LSECs) form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII). Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene (uPA-NOG mice). Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.