Cristina Ionica Øie

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

BackgroundLiver 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.ResultsCultivation 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.ConclusionViability, 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.

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.

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.

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.

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.

Multi-color imaging of sub-mitochondrial structures in living cells using structured illumination microscopy

Abstract The dimensions of mitochondria are close to the diffraction limit of conventional light microscopy techniques, making the complex internal structures of mitochondria unresolvable. In recent years, new fluorescence-based optical imaging techniques have emerged, which allow for optical imaging below the conventional limit, enabling super-resolution (SR). Possibly the most promising SR and diffraction-limited microscopy techniques for live-cell imaging are structured illumination microscopy (SIM) and deconvolution microscopy (DV), respectively. Both SIM and DV are widefield techniques and therefore provide fast-imaging speed as compared to scanning based microscopy techniques. We have exploited the capabilities of three-dimensional (3D) SIM and 3D DV to investigate different sub-mitochondrial structures in living cells: the outer membrane, the intermembrane space, and the matrix. Using different mitochondrial probes, each of these sub-structures was first investigated individually and then in combination. We describe the challenges associated with simultaneous labeling and SR imaging and the optimized labeling protocol and imaging conditions to obtain simultaneous three-color SR imaging of multiple mitochondrial regions in living cells. To investigate both mitochondrial dynamics and structural details in the same cell, the combined usage of DV for long-term time-lapse imaging and 3D SIM for detailed, selected time point analysis was a useful strategy.

High‐affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats

Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high‐affinity VWF binding.

Chip-based optical microscopy for imaging membrane sieve plates of liver scavenger cells

The evanescent field on top of optical waveguides is used to image membrane network and sieve-plates of liver endothelial cells. In waveguide excitation, the evanescent field is dominant only near the surface (~100-150 nm) providing a default optical sectioning by illuminating fluorophores in close proximity to the surface and thus benefiting higher signal-to-noise ratio. The sieve plates of liver sinusoidal endothelial cells are present on the cell membrane, thus near-field waveguide chip-based microscopy configuration is preferred over epi-fluorescence. The waveguide chip is compatible with optical fiber components allowing easy multiplexing to different wavelengths. In this paper, we will discuss the challenges and opportunities provided by integrated optical microscopy for imaging cell membranes.

Optical nanoscopy to reveal structural and functional properties of liver cells (Presentation Recording)

The advent of optical nanoscopy has provided an opportunity to study fundamental properties of nanoscale biological functions, such as liver sinusoidal endothelial cells (LSEC) and their fenestrations. The fenestrations are nano-pores (50-200 nm) on the LSEC plasma membrane that allow free passage of molecules through cells. The fenestrated LSEC also hase a voracious appetite for waste molecules, viruses and nanoparticles. LSEC daily remove huge amounts of waste, nanoparticles and virus from the blood. Pharmaceuticals also need to pass through these fenestrations to be activated (e.g. cholesterol reducing statins) or detoxified by hepatocytes. And, when we age, our LSEC fenestrations become smaller and fewer. Today, we study these cells and structures using either conventional light microscopy on living cells, or high-resolution (but static) methods such as transmission and scanning electron microscopy on fixed (i.e. dead) tissue. Such methods, while very powerful, yield no real time information about the uptake of virus or nanoparticles, nor any information about fenestration dynamics. Therefore, to study LS-SEC, we are now using optical nanoscopy methods, and developing our own, to map their functions in 4 dimensions. Attaining this goal will shed new light on the cell biology of the liver and how it keeps us alive. This paper describes the challenges of studying LS-SEC with light microscopy, as well as current and potential solutions to this challenge using optical nanoscopy.