1 ifad2017 bernard van den berg - visualising glycocalix
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Considerations for visualizing the glycocalyx: Functional- or structural properties laborious experimental procedures (electron microscopy) in vivo, -vitro models Degradation (biomarker) products some techniques and biomarkers already available to study in human no conclusive markers yet Composition through immunohistochemistry Possible for paraffin sections, useful for pathologic samples Direct visualization of luminal glycocalyx not always possible Indirect visualization possible via extracellular matrix changes
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1 ifad2017 bernard van den berg - visualising glycocalix
- 1. Structure and function Visualizing the endothelial glycocalyx Bernard van den Berg IFAD WORKSHOP 2, NOVEMBER 23TH The Einthoven Laboratory for Vascular and Regenerative Medicine – Division of Nephrology
- 2. What is a glycocalyx? 28-Nov-172 Cells protect themselves from the environment through a gel-like coat This layer also facilitates cell communication with the environment Bacterial B. subtilis glycocalyx Endothelial glycocalyx glyco = sweet, calyx = husk (shell) Allon Weiner, The Weizmann Institute of Science, Rehovot, Israel. 2006. van den Berg, et al. Circ Res 2003 & Endothelial Biomedicine ed., W.C. Aird, 2007 IFAD meeting 2017
- 3. Other surface glycocalyces 28-Nov-173 Intestinal “mucosal” glycocalyx Schürch, et al. Biochimica et Biophysica Acta 1998 Junqueira & Carneiro Basic Histology 11th ed., McGraw-Hill, 2005 Lung alveolar surface film IFAD meeting 2017
- 4. Endothelial glycocalyx involvement 28-Nov-174 IFAD meeting 2017 Endothelial function Permeability Coagulation Inflammation Dane, et al., Am J Physiol 2015
- 5. Cell surface composition 28-Nov-175 Fuster & Esko, Nature reviews, 2005 Proteoglycans Glycosaminoglycans Hyaluronan IFAD meeting 2017 Heparan sulphate
- 6. Glomerular endothelial glycocalyx 28-Nov-176 Hyaluronan Thrombomodulin Syndecan-1 Syndecan-4 Perlecan Glypican-1 Glycocalyx EC GBM Podocytic foot processes Dane, et al. Am J Physiol 2015 Luminal capillary glycosaminoglycan distribution (LEA, green) Endothelial marker (CD31, red; merged signal is white) Second harmonic signal (blue) IFAD meeting 2017
- 7. Visualizing the endothelial glycocalyx 28-Nov-177 IFAD meeting 2017 High molecular weight fluorescent tracers (functional properties) Electron microscopy (structural properties) Biochemical determination (degradation products) Immunohistochemistry (composition)
- 8. Plasma molecules are confined to lumen 28-Nov-178 IFAD meeting 2017 Bright field Fluorescence 4.7 mm5.4 mm Capillary diameter Exclusion zone (Endothelial surface layer) Red blood cell (RBC) Fluorescent dye (FITC-dextran 70) White blood cell (WBC) Vink & Duling, Circulation Research 1996
- 9. Barrier function: size depends 28-Nov-179 IFAD meeting 2017 T0 min Filling with FITC-dextran T30 min Radial movement of dextran molecules EC labeled with “DiI” Van Haaren, et al. Am J Physiol 2003 Glycocalyx Vessel wall Cannulated artery
- 10. Systemic glycocalyx volume determination 28-Nov-1710 IFAD meeting 2017 ApoE-KO 14 wks, no diet (n=6) Meuwese, et al. PLosOne 2010 ApoE-KO, 10 weeks HFC & 4 weeks hyaluronidase Zuurbier, et al. J Applied Physiol 2005 Van Teeffelen, et al. Hypertension 2007 Tracer dilution technique Tracer mix (100μL): • CFSE-labeled erythrocytes • Dextrans (40- and 500 kDa) • Ficoll (400 kDa) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 1 2 Volume(mL) Hyaluronidase (10U/hr) Vtotal Vglycocalyx Vplasma Vrbc - +
- 11. Barrier function: glycocalyx is a fixed-charge layer 28-Nov-1711 IFAD meeting 2017 Van den Berg BM, Nieuwdorp M, Stroes S & Vink H, 2007 Endothelial Biomedicine. Editor W.C. Aird Part II: Endothelial cell as input-output device, coupling 3D microscopic reconstruction of fluorescent tracer distributions 40kDa Sulfated Dextran (anionic) 40kDa Texas Red Dextran (neutral) Glycocalyx volume (TR40 –S40)
- 12. In vivo infusion of LDL-bodipy 28-Nov-1712 IFAD meeting 2017 Common carotid Carotid sinus Subendothelial space Endothelium Blood vessel lumen van den Berg, et al. Pflugers Arch – Eur J Physiol 2009
- 13. Adherentleukocytes/100µm Endothelial glycocalyx and injury 28-Nov-1713 Constantinescu, et al. ATVB 2003 Perfusion of a venule with heparan sulfates attenuates Ox-LDL mediated Leukocyte immobilization IFAD meeting 2017
- 14. Visualizing the endothelial glycocalyx 28-Nov-1714 IFAD meeting 2017 High molecular weight fluorescent tracers (functional properties) Electron microscopy (structural properties) Biochemical determination (degradation products) Immunohistochemistry (composition)
- 15. First demonstration of endothelial glycocalyx by TEM 28-Nov-1715 IFAD meeting 2017 Luft J, Federation Proceedings 1966 100 nm Cationic dye (Ruthenium red) staining of mouse diaphragmatic capillary
- 16. Different perfusion strategy to preserve carbohydrates 28-Nov-1716 IFAD meeting 2017 Rostgaard & Qvortrup Microvascular Research 1997 Perfusion fixation with: 1. Fluorocarbon (13.3%) /glutaraldehyde (2%) in 0.05 M sodium phosphate buffer, pH 7.4. 2. Glutaraldehyde(2%) in 0.05 M sodium cacodylate buffer (pH 7.4). On ice 3. Postfixation in OsO4 (1%) in 0.12 M cacodylate buffer (pH 7.4) 4. Tannic acid (1%) in distilled water 5. Uranyl acetate (1%) in distilled water 1.0 µm 0.5 µm
- 17. New approach to stabilize carbohydrates for TEM 28-Nov-1717 IFAD meeting 2017 Alcian blue 8GX R = SO2-NH(CH2)3-N(CH3)2 R2-N R2-N R2-N R2-N Cu N-R2 N-R2 N-R2 N-R2 Hyaluronan Perfusion with a cardioplegic salt solution containing 0.1% albumin at a constant flow (pressure monitored) Perfusion fixation/staining with 0.05% Alcian Blue 8GX in a 84mM phosphate buffered 4% paraformaldehyde, 1% glutaraldehyde fixative (pH 7.4) containing 30mM of MgCl2 Post-fixation with a 1% aqueous OsO4 and 1% Lanthanum nitrate solution (Behnke and Zelander, 1970 and Shea, 1971) En-bloc staining with a 1% aqueous Uranyl acetate solution. Contrasting with Reynolds lead citrate.
- 18. New approaches to stabilize carbohydrates for TEM 28-Nov-1718 IFAD meeting 2017 Myocardial capillary van den Berg, et al. Circ Res 2003 Perfusion fixation with Alcian blue 8GX Perfusion fixation with Lanthanium nitrate Chappell, et al. Shock 2010 Myocardial capillary 0.1µm Perfusion fixation with Cupromeronic blue Glomerular capillary Dane, et al., Am J Pathol 2013 Boels, et al. Diabetes 2016 0.1µm Perfusion with cationic ferritin 0.2µm
- 19. Combination of chemical fixation and high pressure freezing (HPF) for TEM 28-Nov-1719 IFAD meeting 2017 VanHecke, et al. Methods in Cell Biology 2008Sosinsky, et al J Struct Biol 2008 aldehyde fixation aldehyde fixation & HPF Immersion fixation Freeze substitution Work flowTissue sample (1 x 0.15 mm) HPFvibratome & Ultra- microtome TEM plasticembedding
- 20. HPF and FS of kidney for TEM 28-Nov-1720 IFAD meeting 2017 High pressure frozen mouse glomerulus stained during freeze substitution with 0.05% acridine orange & 0.2% uranyl acetate in anhydrous acetone 0.5 µm 0.2 µm 0.5 µm 0.2 µm Dane, et al. Am J Physiol 2015
- 21. Visualizing the endothelial glycocalyx 28-Nov-1721 IFAD meeting 2017 High molecular weight fluorescent tracers (functional properties) Electron microscopy (structural properties) Biochemical determination (degradation products) Immunohistochemistry (composition)
- 22. Mechanism of glycocalyx degradation 28-Nov-1722 IFAD meeting 2017 Rabelink & de Zeeuw Nat Rev Nephrol 2015
- 23. Determination of proteoglycan shedding 28-Nov-1723 IFAD meeting 2017 Microchip nanoLC-MS system for glycoprofiling Dane, et al. Clin J Am Soc Nephrol 2014 Thrombomodulin is an anticoagulant cell surface proteoglycan, specific for endothelial cells, and shed upon inflammation Syndecan 1 is major heparan sulfate proteoglycan on endothelial surface, and shed upon inflammation
- 24. Heparan sulfate (HS) biosynthesis and regulation 28-Nov-1724 Rabelink, et al. Nat Rev Nephrol 2017 Heparanase (HPSE1) endo-β(1–4)-D-glucuronidase activity IFAD meeting 2017
- 25. Determination of HPSE degraded HS composition 28-Nov-1725 IFAD meeting 2017 Microchip nanoLC-MS system for glycoprofiling GLYCOREN & GLYCOTREAT consortia together with Radboud UMC (Nijmegen) and Maastricht University (Maastricht), the Netherlands 3S NS FGFR AT-III O-Ser 2S 2S 6S NS 6S NS 6S NS 6S NS 6S NS α4 α4β4 α4 α4 β4β4 6S NS α4 α4 β4 α4 α4 α4 α4α4 α4β4 β4α4 α4 α4 α4α4 α4β4 β4β3 β3 2S 2S FGF = D-Glucuronic acid = L-Iduronic acid = D-Galactose = D-Xylose = N-Acetyl glucosamine HPSEHPSE
- 26. Visualizing the endothelial glycocalyx 28-Nov-1726 IFAD meeting 2017 High molecular weight fluorescent tracers (functional properties) Electron microscopy (structural properties) Biochemical determination (degradation products) Immunohistochemistry (composition)
- 27. Detection of composition using lectins 28-Nov-1727 IFAD meeting 2017 Dane, et al. Am J Pathol 2013 WGA LEA LEA-FITC - Lycopersicon esculentum -(1,4)-linked N-acetyl-glucosamine WGA-TRITC - Triticum vulgaris N-acetylneuraminic acid, N-acetyl--D-glucosamine
- 28. Detection of HS compositional changes 28-Nov-1728 IFAD meeting 2017 10E4 JM403 AO4B08 EW4G2 HS4C3 Domain specific antibodies 10E4 GlcA-GlcNS-GlcA-GlcNAc N-sulfated/N-acetylated HS domain JM403 GlcN GlcUA-rich sequences with N-unsubstituted GlcN units AO4B08 GlcNS6S-IdoA2S N- and 6-O-sulfated HS domains EW4G2 GlcNS6S-GlcA N- and 6-O-sulfated HS domains HS4C3 GlcNS3S6S-GlcA/IdoA2S N- and/or 2-O-sulfated HS domains Lensen, et al. J Am Soc Nephrol 2005 Rops, et al. Nephrol Dial Transplant 2007 Carbohydrate moieties required for antibody binding Antibody Preferred chemical group
- 29. Heparan sulfate specific antibodies 28-Nov-1729 Boels, et al. Diabetes 2016 EC pericytes Normal human serum Diabetic human serum WGA 10E4 WGA/10E4/Hoechst IFAD meeting 2017
- 30. Heparan sulfate specific antibodies 28-Nov-1730 Boels, et al. Am J Pathol 2017 CS56 Con Diab anti-Chondroitin sulfate Control Diabetes JM403 10E4 Control Diabetes Con Diab Con Diab anti-Heparan sulfate IFAD meeting 2017
- 31. Summary 28-Nov-1731 Considerations for visualizing the glycocalyx: Functional- or structural properties laborious experimental procedures (electron microscopy) in vivo, -vitro models Degradation (biomarker) products some techniques and biomarkers already available to study in human no conclusive markers yet Composition through immunohistochemistry Possible for paraffin sections, useful for pathologic samples Direct visualization of luminal glycocalyx not always possible Indirect visualization possible via extracellular matrix changes IFAD meeting 2017
- 32. 28-Nov-1732 Acknowledgements Leiden University Medical Center Internal Medicine Martijn Dane, Margien Boels, Gangqi Wang, Wendy Sol, Angela Koudijs, Ton Rabelink MCB – Electron Microscopy Cristina Avramut, Bram Koster Academic Medical Center, Amsterdam Medical Physics Alina Constantinescu, Paul van Haaren, Jurgen van Teeffelen, Ed van Bavel, Jos Spaan, Hans Vink Vascular Medicine Marijn Meuwese, Max Nieuwdorp, Erik Stroes Electron Microscopy Henk van Veen, Jan van Marle Maastricht University Physiology Dan Potter, Hanneke Cobbelens, Mayella Kuikhoven Biomedical Engineering Sietze Reitsma, Marc van Zandvoort IFAD meeting 2017
Editor's Notes
- The endothelium is part of the glomerular permeability barrier, which consist further of the GBM and podocytes, which with their foot processes reach around the glomerular capillaries. While especially the fenestrae may facilitate the formation of high volumes of ultra-filtrate, their diameters of about 60 to 80nm would also imply loss of macromolecules such as albumin, the endothelial surface is covered with a polysaccharide rich protein gel-like structure, the glycocalyx, or endothelial surface layer (ESL) spanning both the fenestrated- and inter fenestrated domains. The glycocalyx forms a biologically very active surface layer, which may stretch out up to 500 nm into the vascular lumen. The Bacterium Bacillus subtilis taken with a Tecnai T-12 TEM. Taken by Allon Weiner, The Weizmann Institute of Science, Rehovot, Israel. 2006. Microvilli are apical extensions of the cell that are filled with actin. An extracellular coat (glycocalyx) is bound to the plasmalemma of the microvilli. The terminal web is a network that contains actin filaments, intermediate filaments and spectrin. Junqueira, LC and Carneiro, J, Basic Histology 11th ed., McGraw-Hill, New York, 2005. P. 72.
- Transmission electron micrograph from guinea pig lung after fixation with non-aqueous osmium perfluorocarbon mixture. The surfactant film (arrows) is preserved and continuous and overlies a thin hypophase which is thicker at some sites (AL) above the type I epithelium (Ep). End, endothelial cell ; BM, basement membrane. Bar= 0.5 µm. Microvilli are apical extensions of the cell that are filled with actin. An extracellular coat (glycocalyx) is bound to the plasmalemma of the microvilli. The terminal web is a network that contains actin filaments, intermediate filaments and spectrin. Junqueira, LC and Carneiro, J, Basic Histology 11th ed., McGraw-Hill, New York, 2005. P. 72.
- Schematic overview of the endothelial glycocalyx (EG) under healthy and diseased conditions. Left: in a physiological state, the EG protects against protein leakage, inflammation, and coagulation. Heparan sulfates, bound to a heparan sulfate (HS) core protein, and hyaluronan (HA), bound to e.g., CD44, are the main constituents of the endothelial glycocalyx. The order and modification of disaccharide repeats within HS determine the binding site for specific proteins. Right: upon endothelial activation, heparan sulfate disaccharide modification occurs, resulting in a change in protein binding sites. During a chronic disease condition, the EG gets damaged, mainly due to upregulation of glycocalyx-degrading enzymes such as hyaluronidase, heparanase, and proteinases. Shed proteoglycans and glycocalyx fragments in the serum can bind and influence circulating leukocytes. Both HS modification and EG degradation result in inflammation, coagulation, and protein leakage.
- The endothelium is part of the glomerular permeability barrier, which consist further of the GBM and podocytes, which with their foot processes reach around the glomerular capillaries. While especially the fenestrae may facilitate the formation of high volumes of ultra-filtrate, their diameters of about 60 to 80nm would also imply loss of macromolecules such as albumin, the endothelial surface is covered with a polysaccharide rich protein gel-like structure, the glycocalyx, or endothelial surface layer (ESL) spanning both the fenestrated- and inter fenestrated domains. The glycocalyx forms a biologically very active surface layer, which may stretch out up to 500 nm into the vascular lumen.
- We demonstrated by direct observation that FITC-Dex148 is excluded from a 2-to 3-µm-thick region luminal of the endothelium in isolated small arteries. In contrast, FITC-Dex50 and FITC-Dex4 were able to penetrate this region, and the latter also penetrated the arterial wall.
- FIG. 1. (a) Electron micrograph of a fenestrated capillary from the propria of the small intestine processed using procedure 1. The fenestrae are closed by a thin diaphragm (arrows). Note the ‘‘empty’’ area just below the marked diaphragms and compare with Figs. 3f and 6b; capillary lumen (L), basal lamina (BL). Bar: 0.5 mm. (b and c) Electron micrographs of fenestrated capillary from the propria of the small intestine processed using procedure 2. In each fenestra a bush-like structure (filamentous plug) is visible (arrows). Some ‘‘bushes’’ appear unrelated to fenestrae a phenomenon to be expected in sectioned material because ‘‘bushes’’ are larger than fenestrae; capillary lumen (L). Bars: (b) 1.0 µm; (c) 0.5 µm. (d) Tangential section. Each plug (arrows) is composed of 20–40 filamentous molecules (arrowheads). Each filament is about 5–10 nm thick; capillary lumen (L). Bar: 0.1 mm.
- EG stained in a glomerular capillary of a high-pressure frozen kidney section. EG was stained afterward with acridine orange and uranyl acetate during the freeze substitution stage. Shown are an overview (left) and detailed image of the glycocalyx on top of the glomerular filtration barrier (right). EC, endothelial cell; GBM, glomerular basement membrane; P, podocytes; Glx, glycocalyx. Scale bars 500 nm (left) and 200 nm (right).
- Mechanism of glycocalyx degradation. Proheparanase is released by activated endothelial cells and activated platelets and by secretion from leucocytes. Proheparanase is cleaved into active heparanase by cathepsin L. Proteoglycans such as syndecan‑1, but also LDL receptor-related proteins and mannose 6‑phosphate receptors, facilitate recapture of proheparanase by the leucocytes. Proteolysis occurs either in the plasma as a result of cathepsin secretion from monocytes, or in the late endosomes and lysosomes of macrophages following internalization of proheparanase. After internal proteolysis, activated heparanase is secreted alongside exosomes, which also contain enzymes involved in extracellular matrix remodelling. Heparanase consequently cleaves heparan sulfate in the glycocalyx. Released hyaluron and heparan sulfate fragments promote inflammation. Remodelling of the glycocalyx facilitates endothelium–leucocyte interaction. Altogether, this process enables serum proteins, such as albumin and lipoproteins, to enter the subendothelial space.
- Measurements of circulatory endothelial and endothelial surface layer damage markers in participants with and without loss of renal function. Markers were measured in healthy controls (n=10) and patients with ESRD (n=23), stable KTx (n=12), and IFTA (n=10). Serum soluble thrombomodulin (sTM). Plasma shed syndecan-1. Box plot whiskers indicate 1st and 99th percentiles. *P,0.05; **P,0.01; **P,0.001 TM is an anticoagulant cell surface proteoglycan that is shed from the endothelial cell surface layer after inflammatory stimulation, resulting in the sTM that we measured in serum (30). Although it is also expressed in low amounts by dendritic cells and monocytes, it is mainly expressed by endothelial cells, thereby making it a reasonable marker for shedding of proteoglycans from the ESL. Syndecan-1 is also shown to be expressed at the luminal endothelial surface (32,33). Its shedding from the ESL under inflammatory conditions is thought to contribute to plasma levels of soluble syndecan- 1 (34). In agreement, syndecan-1 shedding has been shown
- Heparanase structure and binding of heparan sulfate to its cleavage site. a | Electrostatic surface projection of the 3D crystallographic structure of heparanase (PDB code 5E8M from the RSCB Protein Data Bank) with negative (red), neutral (white) and positive (blue) charged sites (modelled with UCSF Chimera software). The potential domains that can bind the negatively charged heparan sulfate (HS) are indicated as HS binding domain (HSBD) 1 and HSBD2 with the HS-binding cleft in between. Monoclonal antibodies raised against the amino acid motif Lys158–Asn162 in HSBD1 have heparanase neutralizing properties. b | Model of HS showing the tetrasaccharide repeat of glucuronic acid (GlcA) and N,6‑O‑sulfated glucosamine (GlcNS/6S) at which heparanase (HPSE) cuts from the non-reducing end. The magnified image shows how HS is predicted to bind the heparanase active domain, especially to negatively charged sulfate domains on the two N,6‑O‑sulfated glucosamines.
- Kidney tissue was dissected into sections (100µm) with a vibratome Tissue slices were washed twice with HBSS containing 0.5% BSA, 5 mmol/L HEPES, and 0.03 mmol/L EDTA (HBSS-BSA). Slices were stained overnight with 10 µg/mL of various fluorescently labeled lectins Slices in HBSS-BSA were fixed to the bottom of a Petri dish and were examined using a CLSM (710-NLO; Carl Zeiss, Göttingen, Germany) and a 40x objective lens (Plan Neo Fluar NA 1.3/oil differential interference contrast; Carl Zeiss). Confocal 12-bit gray-scale axial images (xy dimensions, 100x100 µm) of the glomerulus were recorded using ZEN-2009 Image software