Protocol for Isolation and Culture of Mouse Hepatocytes (HCs), Kupffer Cells (KCs), and Liver Sinusoidal Endothelial Cells (LSECs) in Analyses of Hepatic Drug Distribution

Development of the new generation of drugs (e.g., oligo- and polynucleotides administered intravascularly either as free compounds or as nano-formulations) frequently encounters major challenges such as lack of control of targeting and/or delivery. Uncontrolled or unwanted clearance by the liver is a well-known and particularly important hurdle in this respect. Hence, reliable techniques are needed to identify the type(s) of liver cells, receptors, and metabolic mechanisms that are responsible for unwanted clearance of these compounds. We describe here a method for the isolation and culture of the major cell types from mouse liver: hepatocytes (HCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs). The presently described protocol employs perfusion of the liver with a collagenase-based enzyme preparation to effectively transform the intact liver to a single cell suspension. From this initial cell suspension HCs are isolated by specified centrifugation schemes, yielding highly pure HC preparations, and KCs and LSECs are isolated by employing magnetic-activated cell sorting (MACS). The MACS protocol makes use of magnetic microbeads conjugated with specific antibodies that bind unique surface antigens on either KCs or LSECs. In this way the two cell types are specifically and separately pulled out of the initial liver cell suspension by applying a magnetic field, resulting in high purity, yield, and viability of the two cell types, allowing functional studies of the cells. If the drug compound in question is to be studied with respect to liver cell distribution of intravascularly administered drug compounds the isolated cells can be analyzed directly after isolation. Detailed studies of receptor-ligand interactions and/or dynamics of intracellular metabolism of the compound can be conducted in primary surface cultures of HCs, LSECs, and KCs established by seeding the isolated cells on specified growth substrates

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Scavenger properties of cultivated pig liver endothelial cells

BACKGROUND: The liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC. RESULTS: Endocytosis of five (125)I-labelled molecules: collagen α-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of α-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells. CONCLUSION: Pig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC

The scavenger function of liver sinusoidal endothelial cells in health and disease

The aim of this review is to give an outline of the blood clearance function of the liver sinusoidal endothelial cells (LSECs) in health and disease. Lining the hundreds of millions of hepatic sinusoids in the human liver the LSECs are perfectly located to survey the constituents of the blood. These cells are equipped with high-affinity receptors and an intracellular vesicle transport apparatus, enabling a remarkably efficient machinery for removal of large molecules and nanoparticles from the blood, thus contributing importantly to maintain blood and tissue homeostasis. We describe here central aspects of LSEC signature receptors that enable the cells to recognize and internalize blood-borne waste macromolecules at great speed and high capacity. Notably, this blood clearance system is a silent process, in the sense that it usually neither requires or elicits cell activation or immune responses. Most of our knowledge about LSECs arises from studies in animals, of which mouse and rat make up the great majority, and some species differences relevant for extrapolating from animal models to human are discussed. In the last part of the review, we discuss comparative aspects of the LSEC scavenger functions and specialized scavenger endothelial cells (SECs) in other vascular beds and in different vertebrate classes. In conclusion, the activity of LSECs and other SECs prevent exposure of a great number of waste products to the immune system, and molecules with noxious biological activities are effectively “silenced” by the rapid clearance in LSECs. An undesired consequence of this avid scavenging system is unwanted uptake of nanomedicines and biologics in the cells. As the development of this new generation of therapeutics evolves, there will be a sharp increase in the need to understand the clearance function of LSECs in health and disease. There is still a significant knowledge gap in how the LSEC clearance function is affected in liver disease

Uptake and Degradation of Bacteriophages by Liver Sinusoidal Endothelial Cells

Bacteriophages (briefly, “phages”) are viruses which target bacteria, and are non-infectious to eukaryotic cells. It is estimated that more than 30 billion phages cross into the human body from the gut each day1, and eventually need to be cleared from the blood circulation. The liver plays a central role in pathogen clearance, and liver sinusoidal endothelial cells (LSECs), which form the lining of the numerous capillaries in the liver, are therefore on the front lines for this removal process. However, despite their strategic location and efficiency in removing small (<200 nm) particles2, LSECs have historically been poorly studied in terms of removal of phages. We hypothesized that LSECs play a critical role in the removal of phages from the bloodstream through endocytic uptake and lysosomal degradation, and used GFP-labeled T4 bacteriophages as a model system to study this clearance process. Uptake and trafficking of phages in primary cultured LSECs was monitored by deconvolution microscopy on both short (1 hour) and long (24 hours) term timescales, and structured illumination microscopy was used to confirm the identity of the LSECs using their unique, sub-diffraction scale morphological features: tiny holes called fenestrations. After being taken up by the cells, the phages were rapidly transported to late endosomes/lysosomes, as confirmed by colocalization studies with an LSEC-specific lysosomal vital marker. Challenging the LSEC cultures with radiolabeled phages for up to 24 hours showed that the phages were degraded about 4h after being taken up by the cells, with degradation products being increasingly released to the spent medium up to about 18h after uptake. In conclusion, our novel finding that LSECs internalize and degrade bacteriophages lends support to the hypothesis that LSECs play an important role in the clearance of blood borne phages

Changes in the proteome and secretome of rat liver sinusoidal endothelial cells during early primary culture and effects of dexamethasone

Introduction Liver sinusoidal endothelial cells (LSECs) are specialized fenestrated scavenger endothelial cells involved in the elimination of modified plasma proteins and tissue turnover waste macromolecules from blood. LSECs also participate in liver immune responses. A challenge when studying LSEC biology is the rapid loss of the in vivo phenotype in culture. In this study, we have examined biological processes and pathways affected during early-stage primary culture of rat LSECs and checked for cell responses to the pro-inflammatory cytokine interleukin (IL)-1β and the anti-inflammatory drug dexamethasone. Methods LSECs from male Sprague Dawley rats were cultured on type I collagen in 5% oxygen atmosphere in DMEM with serum-free supplements for 2 and 24 h. Quantitative proteomics using tandem mass tag technology was used to examine proteins in cells and supernatants. Validation was done with qPCR, ELISA, multiplex immunoassay, and caspase 3/7 assay. Cell ultrastructure was examined by scanning electron microscopy, and scavenger function by quantitative endocytosis assays. Results LSECs cultured for 24 h showed a characteristic pro-inflammatory phenotype both in the presence and absence of IL-1β, with upregulation of cellular responses to cytokines and interferon-γ, cell-cell adhesion, and glycolysis, increased expression of fatty acid binding proteins (FABP4, FABP5), and downregulation of several membrane receptors (STAB1, STAB2, LYVE1, CLEC4G) and proteins in pyruvate metabolism, citric acid cycle, fatty acid elongation, amino acid metabolism, and oxidation-reduction processes. Dexamethasone inhibited apoptosis and improved LSEC viability in culture, repressed inflammatory and immune regulatory pathways and secretion of IL-1β and IL-6, and further upregulated FABP4 and FABP5 compared to time-matched controls. The LSEC porosity and endocytic activity were reduced at 24 h both with and without dexamethasone but the dexamethasone-treated cells showed a less stressed phenotype. Conclusion Rat LSECs become activated towards a pro-inflammatory phenotype during early culture. Dexamethasone represses LSEC activation, inhibits apoptosis, and improves cell viability

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIB, yet maintain a preserved fenestration the Glmp gt/gt mouse model of slowly progressing liver fibrosis

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9–10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and agematched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis

pH-dependent structural transitions in cationic ionizable lipid mesophases are critical for lipid nanoparticle function

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m- transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.publishedVersio

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity