Unraveling the transcriptional determinants of liver sinusoidal endothelial cell specialization.

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.

Nanoscopy on-a-chip: super-resolution imaging on the millimeter scale.

Optical nanoscopy techniques can image intracellular structures with high specificity at sub-diffraction limited resolution, bridging the resolution gap between optical microscopy and electron microscopy. So far conventional nanoscopy lacks the ability to generate high throughput data, as the imaged region is small. Photonic chip-based nanoscopy has demonstrated the potential for imaging large areas, but at a lateral resolution of 130 nm. However, all the existing super-resolution methods provide a resolution of 100 nm or better. In this work, chip-based nanoscopy is demonstrated with a resolution of 75 nm over an extraordinarily large area of 0.5 mm × 0.5 mm, using a low magnification and high N.A. objective lens. Furthermore, the performance of chip-based nanoscopy is benchmarked by studying the localization precision and illumination homogeneity for different waveguide widths. The advent of large field-of-view chip-based nanoscopy opens up new routes in diagnostics where high throughput is needed for the detection of non-diffuse disease, or rare events such as the early detection of cancer.

Rat liver sinusoidal endothelial cells (LSECs) express functional low density lipoprotein receptor-related protein-1 (LRP-1).

BACKGROUND & AIMS: The low density lipoprotein receptor-related protein-1 (LRP-1) is a large, multifunctional endocytic receptor from the LDL receptor family, highly expressed in liver parenchymal cells (PCs), neurons, activated astrocytes, and fibroblasts. The aim of the study was to investigate if liver sinusoidal endothelial cells (LSECs), highly specialized scavenger cells, express LRP-1. METHODS: To address this question, experiments were performed in vivo and in vitro to determine if receptor associated protein (RAP) and trypsin-activated α(2)-macroglobulin (α(2)M∗) were endocytosed in LSECs. RESULTS: Both ligands were cleared from the circulation mainly by the liver. Hepatocellular distribution of intravenously administered ligands, assessed after magnetic bead cell separation using LSEC- and KC-specific antibodies, showed that PCs contained 93% and 82% of liver-associated (125)I-RAP and (125)I-α(2)M∗, whereas 5% and 11% were associated with LSECs. Uptake of RAP and α(2)M∗ in the different liver cell population in vitro was specific and followed by degradation. The uptake of (125)I-RAP was not inhibited by ligands to known endocytosis receptors in LSECs, while uptake of (125)I-α(2)M∗ was significantly inhibited by RAP, suggesting the involvement of LRP-1. Immunofluorescence using LRP-1 antibody showed positive staining in LSECs. Ligand blot analyses using total cell proteins and (125)I-RAP followed by mass spectrometry further confirmed and identified LRP-1 in LSECs. CONCLUSIONS: LSECs express functional LRP-1. An important implication of our findings is that LSECs contribute to the rapid removal of blood borne ligands for LRP-1 and may thus play a role in lipid homeostasis.

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies.

The liver as the largest organ in the human body is composed of a complex macroscopic and microscopic architecture that supports its indispensable function to maintain physiological homeostasis. Optical imaging of the human liver is particularly challenging because of the need to cover length scales across 7 orders of magnitude (from the centimeter scale to the nanometer scale) in order to fully assess the ultrastructure of the entire organ down to the subcellular scale and probe its physiological function. This task becomes even more challenging the deeper within the organ one hopes to image, because of the strong absorption and scattering of visible light by the liver. Here, we demonstrate how optical imaging methods utilizing highly specific fluorescent labels, as well as label-free optical methods can seamlessly cover this entire size range in excised, fixed human liver tissue and we exemplify this by reconstructing the biliary tree in three-dimensional space. Imaging of tissue beyond approximately 0.5 mm length requires optical clearing of the human liver. We present the successful use of optical projection tomography and light-sheet fluorescence microscopy to derive information about the liver architecture on the millimeter scale. The intermediate size range is covered using label-free structural and chemically sensitive methods, such as second harmonic generation and coherent anti-Stokes Raman scattering microscopy. Laser-scanning confocal microscopy extends the resolution to the nanoscale, allowing us to ultimately image individual liver sinusoidal endothelial cells and their fenestrations by super-resolution structured illumination microscopy. This allowed us to visualize the human hepatobiliary system in 3D down to the cellular level, which indicates that reticular biliary networks communicate with portal bile ducts via single or a few ductuli. Non-linear optical microscopy enabled us to identify fibrotic regions extending from the portal field to the parenchyma, along with microvesicular steatosis in liver biopsies from an older patient. Lastly, super-resolution microscopy allowed us to visualize and determine the size distribution of fenestrations in human liver sinusoidal endothelial cells for the first time under aqueous conditions. Thus, this proof-of-concept study allows us to demonstrate, how, in combination, these techniques open up a new chapter in liver biopsy analysis.

SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components.

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.

Liver sinusoidal endothelial cells contribute to the uptake and degradation of entero bacterial viruses.

The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.

Photonic-chip assisted correlative light and electron microscopy.

Correlative light and electron microscopy (CLEM) unifies the versatility of light microscopy (LM) with the high resolution of electron microscopy (EM), allowing one to zoom into the complex organization of cells. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) microscopy over large field of view and high precision localization of the target area of interest within EM. The photonic chips are used as a substrate to hold, to illuminate and to provide landmarking of the sample through specially designed grid-like numbering systems. Using this approach, we demonstrate its applicability for tracking the area of interest, imaging the three-dimensional (3D) structural organization of nano-sized morphological features on liver sinusoidal endothelial cells such as fenestrations (trans-cytoplasmic nanopores), and correlating specific endo-lysosomal compartments with its cargo protein upon endocytosis.

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip.

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 µm x 500 µm region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelial cells.

BACKGROUND: Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered in the hepatic sinusoids is in general considerably lower than the oxygen tension in the air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions with regard to oxygen would expect to improve cell survival, structure and function. In this study LSECs were isolated from rats and cultured under either 5% (normoxic) or 20% (hyperoxic) oxygen tensions, and several morpho-functional features were compared. RESULTS: Cultivation of LSECs under normoxia, as opposed to hyperoxia improved the survival of LSECs and scavenger receptor-mediated endocytic activity, reduced the production of the pro-inflammatory mediator, interleukin-6 and increased the production of the anti-inflammatory cytokine, interleukin-10. On the other hand, fenestration, a characteristic feature of LSECs disappeared gradually at the same rate regardless of the oxygen tension. Expression of the cell-adhesion molecule, ICAM-1 at the cell surface was slightly more elevated in cells maintained at hyperoxia. Under normoxia, endogenous generation of hydrogen peroxide was drastically reduced whereas the production of nitric oxide was unaltered. Culture decline in high oxygen-treated cultures was abrogated by administration of catalase, indicating that the toxic effects observed in high oxygen environments is largely caused by endogenous production of hydrogen peroxide. CONCLUSION: Viability, structure and many of the essential functional characteristics of isolated LSECs are clearly better preserved when the cultures are maintained under more physiologic oxygen levels. Endogenous production of hydrogen peroxide is to a large extent responsible for the toxic effects observed in high oxygen environments.

Primary rat LSECs preserve their characteristic phenotype after cryopreservation.

Liver disease is a leading cause of morbidity and mortality worldwide. Recently, the liver non-parenchymal cells have gained increasing attention for their potential role in the development of liver disease. Liver sinusoidal endothelial cells (LSECs), a specialized type of endothelial cells that have unique morphology and function, play a fundamental role in maintaining liver homeostasis. Current protocols for LSEC isolation and cultivation rely on freshly isolated cells which can only be maintained differentiated in culture for a few days. This creates a limitation in the use of LSECs for research and a need for a consistent and reliable source of these cells. To date, no LSEC cryopreservation protocols have been reported that enable LSECs to retain their functional and morphological characteristics upon thawing and culturing. Here, we report a protocol to cryopreserve rat LSECs that, upon thawing, maintain full LSEC-signature features: fenestrations, scavenger receptor expression and endocytic function on par with freshly isolated cells. We have confirmed these features by a combination of biochemical and functional techniques, and super-resolution microscopy. Our findings offer a means to standardize research using LSECs, opening the prospects for designing pharmacological strategies for various liver diseases, and considering LSECs as a therapeutic target.

Quantitative phase microscopy of red blood cells during planar trapping and propulsion.

Red blood cells (RBCs) have the ability to undergo morphological deformations during microcirculation, such as changes in surface area, volume and sphericity. Optical waveguide trapping is suitable for trapping, propelling and deforming large cell populations along the length of the waveguide. Bright field microscopy employed with waveguide trapping does not provide quantitative information about structural changes. Here, we have combined quantitative phase microscopy and waveguide trapping techniques to study changes in RBC morphology during planar trapping and transportation. By using interference microscopy, time-lapsed interferometric images of trapped RBCs were recorded in real-time and subsequently utilized to reconstruct optical phase maps. Quantification of the phase differences before and after trapping enabled study of the mechanical effects during planar trapping. During planar trapping, a decrease in the maximum phase values, an increase in the surface area and a decrease in the volume and sphericity of RBCs were observed. QPM was used to analyze the phase values for two specific regions within RBCs: the annular rim and the central donut. The phase value of the annular rim decreases whereas it increases for the central donut during planar trapping. These changes correspond to a redistribution of cytosol inside the RBC during planar trapping and transportation.

FITC Conjugation Markedly Enhances Hepatic Clearance of N-Formyl Peptides.

In both septic and aseptic inflammation, N-formyl peptides may enter the circulation and induce a systemic inflammatory response syndrome similar to that observed during septic shock. The inflammatory response is brought about by the binding of N-formyl peptide to formyl peptide receptors (FPRs), specific signaling receptors expressed on myeloid as well as non-myeloid cells involved in the inflammatory process. N-formyl peptides conjugated with fluorochromes, such as fluorescein isothiocyanate (FITC) are increasingly experimentally used to identify tissues involved in inflammation. Hypothesizing that the process of FITC-conjugation may transfer formyl peptide to a ligand that is efficiently cleared from the circulation by the natural powerful hepatic scavenging regime we studied the biodistribution of intravenously administered FITC-fNLPNTL (Fluorescein-isothiocyanate- N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys) in mice. Our findings can be summarized as follows: i) In contrast to unconjugated fNLPNTL, FITC-fNLPNTL was rapidly taken up in the liver; ii) Mouse and human liver sinusoidal endothelial cells (LSECs) and hepatocytes express formyl peptide receptor 1 (FRP1) on both mRNA (PCR) and protein (Western blot) levels; iii) Immunohistochemistry showed that mouse and human liver sections expressed FRP1 in LSECs and hepatocytes; and iv) Uptake of FITC-fNLPNTL could be largely blocked in mouse and human hepatocytes by surplus-unconjugated fNLPNTL, thereby suggesting that the hepatocytes in both species recognized FITC-fNLPNTL and fNLPNTL as indistinguishable ligands. This was in contrast to the mouse and human LSECs, in which the uptake of FITC-fNLPNTL was mediated by both FRP1 and a scavenger receptor, specifically expressed on LSECs. Based on these results we conclude that a significant proportion of FITC-fNLPNTL is taken up in LSECs via a scavenger receptor naturally expressed in these cells. This calls for great caution when using FITC-fNLPNTL and other chromogen-conjugated formyl peptides as a probe to identify cells in a liver engaged in inflammation. Moreover, our finding emphasizes the role of the liver as an important neutralizer of otherwise strong inflammatory signals such as formyl peptides.

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.

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells.

BACKGROUND & AIMS: Liver fibrogenesis - scarring of the liver that can lead to cirrhosis and liver cancer - is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. MATERIAL AND METHODS: Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. RESULTS: We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. CONCLUSIONS: Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation.

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.