New insights into the clearance of tissue factor pathway inhibitor (TFPI) and unfractionated heparin (UFH)

Paper number 2 of the thesis is not available in Munin: 2. Oie, C.I., Olsen, R., Smedsrød, S. and Hansen, J.B.: 'Liver sinusoidal endothelial cells are the principal site for elimination of unfractionated heparin from the circulation', American journal of physiology: Gastrointestinal and liver physiology, 2008. 294(2): p. G520-8 (American Physiological Society - publisher's restrictions). Available at http://dx.doi.org/10.1152/ajpgi.00489.2007”Hvor mye?”, ”Hvor ofte?” og ”Hvor lenge?” er sentrale spørsmål i studier av legemidler. For å finne en optimal behandling trenger man kunnskap om et legemiddels opptak, fordeling i kroppen, og nedbryting. Øie sitt prosjekt viser at leveren spiller en nøkkelrolle i nedbrytningen av to sentrale substanser som hemmer blodlevring (antikoagulantia), heparin og tissue factor pathway inhibitor (TFPI). Heparin er det vanligste antikoagulante legemiddel som brukes i pasientbehandling. TFPI produseres i blodåreveggen og utøver sin funksjon ved å hemme blodets levringssystem. Leveren har vist seg å være hovedorganet for omsetning og nedbrytning av en rekke fysiologiske og ikke-fysilogiske substanser i sirkulasjonen. Imidlertid er lite kjent om hvordan leveren er involvert i nedbrytningen av heparin og TFPI. I en dyremodell (Rattus Norvegicus) fant Øie at heparin og TFPI også i hovedsak nedbrytes i leveren, og at det er spesialiserte celler, nemlig leverens sinusoidale endotelceller (LSECs) og hepatocytter som er de ansvarlige cellene i leveren. I årevis har man gjort forsøk på å rense rekombinant TFPI fra menneske som kan brukes som legemiddel for å hemme blodlevring og blodpropp. I sin avhandling viste Øie at TFPI ble fjernet fra kroppen ved forskjellige mekansimer avhengig av om TFPI ble renset fra bakterier eller isolerte celler fra dyr. Et annet unikt funn var at LSECs er ansvarlige for fjerning av heparin fra sirkulasjon. Likeledes fant hun at LSECs også uttrykker en reseptor for binding og nedbrytning av forskjellige substanser som tidligere bare har vært påvist i hepatocytter. Heparin binder seg til en rekke proteiner, inkludert TFPI, i blodet. Dannelse av heparin-TFPI komplekser i sirkulasjonen under heparin behandling kan ha viktige implikasjoner for behandling. I avhandlingen fant Øie at bindingen mellom TFPI og heparin, og nedbrytningen av TFPI-heparin komplekser var avhengig av hvilken type celler TFPI var produsert (bakterie eller dyrceller) i

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

Source at https://doi.org/10.1515/nanoph-2017-0112. Licensed CC BY-NC-ND 4.0.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

Structured Illumination Microscopy of Biological Structures

Abstract of presentation held at Norwegian Electro-Optics Meeting, Henningsvær, Norway, 2-4 May 2018.Resolution in optical microscopy has long been limited to the Abbe diffraction limit, i.e. about 250 nm laterally for visible wavelengths on a very good microscope. In the last two decades several techniques have been devised to circumvent this limit: an achievement which was recognized with the 2014 Nobel Prize in Chemistry. Structured Illumination Microscopy (SIM) was the first of these techniques to become commercially available, and continues to be the only super-resolution technique which is practically compatible with living cells, while also requiring the least modification to conventional sample-labeling protocols. SIM utilizes Moiré patterns and frequency shifting to improve resolution 2X in each dimension, as well as significantly improve the contrast for the mid-range spatial frequencies. These advances have unlocked a new realm of biological inquiry: the combination of the high biochemical specificity of fluorescent probes with resolution previously only possible with electron microscopy now enables the direct study of sub-organelle colocalization and the dynamics of living cells. Here, we will present both the basics of the SIM technique as well as a sampling of its biological applications from our lab at UiT, including sub-mitochondrial localization and dynamics, sieve-like nanostructures in liver cells, and large-scale visualization of super-resolved cardiac tissue sections, as well as discuss the practical limitations and implications of this work

Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study

Liver Sinusoidal Endothelial Cells (LSEC) line the hepatic vasculature providing blood filtration via transmembrane nanopores called fenestrations. These structures are 50−300 nm in diameter, which is below the resolution limit of a conventional light microscopy. To date, there is no standardized method of fenestration image analysis. With this study, we provide and compare three different approaches: manual measurements, a semi-automatic (threshold-based) method, and an automatic method based on user-friendly open source machine learning software. Images were obtained using three super resolution techniques – atomic force microscopy (AFM), scanning electron microscopy (SEM), and structured illumination microscopy (SIM). Parameters describing fenestrations such as diameter, area, roundness, frequency, and porosity were measured. Finally, we studied the user bias by comparison of the data obtained by five different users applying provided analysis methods

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

Photonic-chip assisted correlative light and electron microscopy

Correlative light-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. Most CLEM techniques use ultrathin sections, and thus lack the 3D-EM structural information, and focusing on a very restricted field of view. 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 chip-based direct stochastic optical reconstruction microscopy (dSTORM), and 3D high precision correlation of biological processes by focused ion beam-scanning electron microscopy (FIB-SEM) is further demonstrated. The core layer of the photonic chips are used as a substrate to hold, to illuminate and the cladding layer is used to enable high-precision landmarking of the sample through specially designed grid-like numbering systems. The landmarks are fabricated on the cladding of the photonic chips as extruding pillars from the waveguide surface, thus remaining visible for FIB-SEM after resin embedding during sample processing. Using this approach we demonstrate its applicability for tracking the area of interest, imaging the 3D structural organization of nano-sized morphological features on liver sinusoidal endothelial cells such as fenestrations, and correlating specific endo-lysosomal compartments with its cargo protein upon endocytosis. We envisage that photonic chip equipped with landmarks can be used in the future to automatize the work-flow for both LM and EM for high-throughput CLEM, providing the resolution needed for insights into the complex intracellular communication and the relation between morphology and function in health and disease.Comment: 23 Pages, 11 Figure

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 FITCfNLPNTL 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

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

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