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Nanomechanics of the Endothelial Glycocalyx

From Structure to Function
Open AccessPublished:February 06, 2020DOI:https://doi.org/10.1016/j.ajpath.2019.07.021
      The negatively charged, brush-like glycocalyx covers the surface layer of endothelial cells. This layer of membrane-bound, carbohydrate-rich molecules covers the luminal surface of the endothelium along the entire vascular tree, mostly comprising glycoproteins and proteoglycans. Together with the underlying actin-rich endothelial cortex, 50 to 150 nm beneath the plasma membrane, the endothelial glycocalyx (eGC) is recognized as a vasoprotective nanobarrier and responsive hub. Importantly, both the eGC and cortex are highly dynamic and can adapt their nanomechanical properties (ie, stiffness and height) to changes in the environment. The constant change between a soft and a stiff endothelial surface is imperative for proper functioning of the endothelium. This review defines the nanomechanical properties of the eGC and stresses the underlying mechanisms and factors leading to a disturbed structure–function relationship. Specifically, under inflammatory conditions, the eGC is damaged, resulting in enhanced vascular permeability, tissue edema, augmented leukocyte adhesion, platelet aggregation, and dysregulated vasodilation. An integrated knowledge of the relationship between the nanomechanical properties, structure, and function of the eGC might be key in understanding vascular function and dysfunction. In this context, the clinical aspects for preservation and restoration of proper eGC nanomechanics are discussed, considering the eGC as a potentially promising diagnostic marker and therapeutic target in the near future.
      Living cells are neither rigid nor inflexible but are characterized by high structural dynamics and agility. To fulfill their specific functions, many cell types must be able to easily adapt their nanomechanical properties to the changing requirements and conditions of their habitat. Migrating cancer cells,
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      Actin filament structures in migrating cells.
      erythrocytes, or leukocytes
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      • Piel M.
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      Integrating physical and molecular insights on immune cell migration.
      are prominent examples of structure–function dependencies.
      • Rottner K.
      • Faix J.
      • Bogdan S.
      • Linder S.
      • Kerkhoff E.
      Actin assembly mechanisms at a glance.
      This option to change depends on fast and dynamic alterations in cytoskeletal proteins, such as the actomyosin complex and linker proteins located in a compartment called the cortex, 50 to 150 nm beneath the plasma membrane. Thus, the rigidity, or stiffness, of the cell surface is a mechanical property that reflects the physiological state of cells and can be seen as hallmark for the function of cells.
      • Fels J.
      • Jeggle P.
      • Liashkovich I.
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      Nanomechanics of vascular endothelium.
      ,
      • Kasas S.
      • Dietler G.
      Probing nanomechanical properties from biomolecules to living cells.
      In this context, it is important to mention that the substrate of the cells strongly influences their mechanical properties and thus their function.
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      • Wang Y.L.
      Tissue cells feel and respond to the stiffness of their substrate.
      Thus, cells grown on a stiff substrate are themselves stiffer.
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      • Gooch K.J.
      Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D.
      It should kept in mind that matrix dimensionality might modulate the effects of matrix stiffness on actin and cell stiffness. Thus, efforts have been made to develop models that are similar to the in vivo reality, such as in situ cells derived from ex vivo tissue preparations
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
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      • Maase M.
      • Jeggle P.
      • Martinez-Martinez E.
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      • Loufrani L.
      • Henrion D.
      • Hansen P.B.
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      • Jaisser F.
      The endothelial aENaC participates to vascular endothelial function in vivo.
      • Jeggle P.
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      • Tarjus A.
      • Fassot C.
      • Fels J.
      • Oberleithner H.
      • Jaisser F.
      • Kusche-Vihrog K.
      Epithelial sodium channel stiffens the vascular endothelium in vitro and in Liddle mice.
      or the use of three-dimensional cell culture models.
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      • Yang Y.
      Current state-of-the-art 3D tissue models and their compatibility with live cell imaging.
      For many cell types, a change between soft and stiff is physiologically relevant and tightly linked to specific cellular functions, whereas a loss of this endothelial plasticity might end in cardiovascular pathologies. This review focuses on the surface of endothelial cells: the glycocalyx and the cortical zone beneath the plasma membrane. Together, both layers build a vulnerable zone, able to adapt its nanomechanical properties to any changes of forces exerted by the adjacent bloodstream or provocations conveyed by vasoactive substances. Sustained stiffening of this layer, however, contributes to the development of endothelial dysfunction and vascular pathologies.
      During the past decade, sophisticated methods of investigating the structure of the endothelial glycocalyx (eGC) and its relation to functional demands have been established; this multifunctional surface layer is in focus as both a diagnostic and a therapeutic target. The structure–function relationship of the eGC is strongly associated with diseases related to the cardiovascular system. Recently, eGC in vivo measurements were established, enabling the assessment of eGC nanomechanics (ie, thickness) in the microcirculatory system of critically ill patients. Thus, the focus of the present review is the link between in vitro investigations of the nanomechanical properties of the eGC and its clinical relevance in vivo, highlighting eGC degradation during vascular inflammation.

      Structure and Composition of the eGC

      The surface of endothelial cells is built of different layers that together act as a biological interface between the lumen of blood vessels and the cytoplasm. This arrangement enables coordinated and complex reactions to diverse stimuli coming with the streaming blood. During the past few years, two distinct and mechanically distinguishable compartments have been identified at the cell surface: the glycocalyx and the cortex, including plasma membrane and submembranous cytoskeletal proteins.
      • Kasas S.
      • Wang X.
      • Hirling H.
      • Marsault R.
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      • Yersin A.
      • Regazzi R.
      • Grenningloh G.
      • Riederer B.
      • Forro L.
      • Dietler G.
      • Catsicas S.
      Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly.
      • Weinbaum S.
      • Tarbell J.M.
      • Damiano E.R.
      The structure and function of the endothelial glycocalyx layer.
      • Oberleithner H.
      • Callies C.
      • Kusche-Vihrog K.
      • Schillers H.
      • Shahin V.
      • Riethmuller C.
      • MacGregor G.A.
      • de Wardener H.E.
      Potassium softens vascular endothelium and increases nitric oxide release.
      • Oberleithner H.
      • Peters W.
      • Kusche-Vihrog K.
      • Korte S.
      • Schillers H.
      • Kliche K.
      • Oberleithner K.
      Salt overload damages the glycocalyx sodium barrier of vascular endothelium.
      The eGC is described as the top surface layer of endothelial cells. It is a negatively charged, brush-like layer of membrane-bound, carbohydrate-rich molecules covering the luminal surface of the endothelium along the entire vascular tree.
      • Weinbaum S.
      • Tarbell J.M.
      • Damiano E.R.
      The structure and function of the endothelial glycocalyx layer.
      This network comprises mostly glycoproteins and proteoglycans. The glycoproteins are characterized by short, carbohydrate side chains capped with sialic acid, while the proteoglycans are decorated with long, unbranched glycosaminoglycans. The glycosaminoglycans are composed of heparan sulfate (HS; 50% to 90%), chondroitin sulfate, and hyaluronic acid.
      • Sarrazin S.
      • Lamanna W.C.
      • Esko J.D.
      Heparan sulfate proteoglycans.
      ,
      • Tkachenko E.
      • Rhodes J.M.
      • Simons M.
      Syndecans: new kids on the signaling block.
      Both glycoproteins and proteoglycans consist of core proteins to which one or more sugar side chains are covalently anchored. Transmembrane syndecans and the membrane-bound glypicans are the major protein core families found on the endothelial cell plasma membrane.
      • Reitsma S.
      • Slaaf D.W.
      • Vink H.
      • van Zandvoort M.A.
      • Oude Egbrink M.G.
      The endothelial glycocalyx: composition, functions, and visualization.
      Importantly, with this anchor between the eGC and the endothelial cell cortex, the transduction of biochemical and biomechanical signals from the intravascular compartment toward endothelial cells is enabled, making it a crucial interface between the blood and vascular wall.
      • Weinbaum S.
      • Zhang X.
      • Han Y.
      • Vink H.
      • Cowin S.C.
      Mechanotransduction and flow across the endothelial glycocalyx.
      The cytoplasmic tail of syndecans, for example, associates with the cortical cytoskeleton through linker molecules such as ezrin, tubulin, syntenin, syndesmos, dynamin, and α-actinin and is thus able to distribute force throughout the cell.
      • Weinbaum S.
      • Tarbell J.M.
      • Damiano E.R.
      The structure and function of the endothelial glycocalyx layer.
      The mutual interference between the eGC and endothelial cortex, in particular, is discussed below (Mutual Interference between Glycocalyx and Endothelial Cortex). The dynamic interactions between those molecules, however, determine the mechanical properties and thus the function of the respective region.
      As reported, the functional thickness of the eGC varies from a few hundred nanometers in capillaries to a few micrometers in arteries.
      • Nieuwdorp M.
      • Meuwese M.C.
      • Vink H.
      • Hoekstra J.B.
      • Kastelein J.J.
      • Stroes E.S.
      The endothelial glycocalyx: a potential barrier between health and vascular disease.
      This obvious discrepancy might be due to the technical limitations of measuring the glycocalyx at the surface of a variety of cells or the fact that the enormous flexibility is one of the most important features of the structure. In this context it is important to mention that changes in the nanomechanical properties (ie, stiffness and functional thickness) of the eGC alter its function.
      • Fels J.
      • Jeggle P.
      • Liashkovich I.
      • Peters W.
      • Oberleithner H.
      Nanomechanics of vascular endothelium.
      For a long time, measuring the functional thickness or height of the eGC was delicate and a technical challenge. New techniques for determining the height of the eGC have been developed. By stabilizing the anionic carbohydrate structures of the eGC, a thickness of up to 0.5 μm was demonstrated in rat left ventricular myocardial capillaries.
      • van den Berg B.M.
      • Vink H.
      • Spaan J.A.
      The endothelial glycocalyx protects against myocardial edema.
      By direct in vivo measurements of the eGC in hamster cremaster muscle, Vink and Duling
      • Vink H.
      • Duling B.R.
      Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries.
      provided evidence for an eGC thickness of 0.4 to 0.5 μm, which was supported by recent data derived from intravital microscopy.
      • Gao L.
      • Lipowsky H.H.
      Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes.
      ,
      • Dane M.J.
      • van den Berg B.M.
      • Lee D.H.
      • Boels M.G.
      • Tiemeier G.L.
      • Avramut M.C.
      • van Zonneveld A.J.
      • van der Vlag J.
      • Vink H.
      • Rabelink T.J.
      A microscopic view on the renal endothelial glycocalyx.
      Using techniques for measuring the permeability to small solutes, it was suggested that specific components are distributed within the eGC in a non-uniform manner
      • Gao L.
      • Lipowsky H.H.
      Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes.
      In particular, two different layers within the eGC could be identified, each consisting of typical glycosaminoglycans: a layer at the luminal side of the eGC (thickness, approximately 460 nm, consisting of mainly HS) and a denser, endothelial cell–adjacent sublayer (thickness, approximately 200 nm, consisting mainly of chondroitin sulfate and hyaluronic acid).
      • Gao L.
      • Lipowsky H.H.
      Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes.
      Thus, the eGC itself is a multilayered structure containing layers of different composition and mechanical/functional properties.
      • Peters W.
      • Drueppel V.
      • Kusche-Vihrog K.
      • Schubert C.
      • Oberleithner H.
      Nanomechanics and sodium permeability of endothelial surface layer modulated by hawthorn extract WS 1442.
      Removing the HS enzymatically, however, results in decreases in eGC height and stiffness (Figure 1, A and B ).
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      Figure thumbnail gr1
      Figure 1Effects of enzyme digestion, proinflammatory agents, and shear stress on the nanomechanical properties and integrity of the eGC. Using atomic force microscope–based nanoindentation measurements, the height and stiffness of the eGC were determined. A and B: Enzymatic removal of heparan sulfate (Hep) residues by heparinase I (1 SU/mL) led to reductions in eGC height (A) and stiffness (B) of endothelial cells. C and D: Treatment with tumor necrosis factor (TNF)-α (10 ng/mL) for 60 minutes decreased eGC height (C) and stiffness (D) of human pulmonary microvascular endothelial cells. Data are expressed as means ± SEM. n = 3 to 5. ∗P < 0.05. Data in B and D were modified from Wiesinger et al.
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.

      Probing the Nanomechanical Properties of the Glycocalyx

      The eGC is a fragile and highly vulnerable scaffold that complicates its observation and analysis. Fixation, dehydration, and staining procedures have adverse effects that hamper a proper examination of the eGC structure. Pioneered by Luft
      • Luft J.H.
      Fine structures of capillary and endocapillary layer as revealed by ruthenium red.
      >50 years ago, the presence of the eGC in the lumen of vessels has been successfully shown using transmission electron microscopy, followed by the development of variations in staining and preservation techniques.
      • Becker B.F.
      • Chappell D.
      • Jacob M.
      Endothelial glycocalyx and coronary vascular permeability: the fringe benefit.
      • Chappell D.
      • Jacob M.
      • Paul O.
      • Rehm M.
      • Welsch U.
      • Stoeckelhuber M.
      • Conzen P.
      • Becker B.F.
      The glycocalyx of the human umbilical vein endothelial cell: an impressive structure ex vivo but not in culture.
      • Faas F.G.
      • Avramut M.C.
      • van den Berg B.M.
      • Mommaas A.M.
      • Koster A.J.
      • Ravelli R.B.
      Virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps.
      Using specific antibodies against carbohydrate domains within HS, hyaluronic acid, or chondroitin sulfate, or against proteoglycans or fluorescently labeled lectins (lycopersicon esculentum agglutinin or wheat germ agglutinin), the eGC can be imaged with fluorescence microscopy. With confocal and two-photon microscopy, the optical spatial resolution is high enough to obtain detailed information about the eGC structure and composition.
      • Vogel J.
      • Sperandio M.
      • Pries A.R.
      • Linderkamp O.
      • Gaehtgens P.
      • Kuschinsky W.
      Influence of the endothelial glycocalyx on cerebral blood flow in mice.
      • Salmon A.H.
      • Satchell S.C.
      Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.
      • Betteridge K.B.
      • Arkill K.P.
      • Neal C.R.
      • Harper S.J.
      • Foster R.R.
      • Satchell S.C.
      • Bates D.O.
      • Salmon A.H.
      Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function.
      However, from all studies, it has become clear that an exact and uninfluenced quantification of the eGC is possible only with living, untreated cells in vitro or ex vivo. Thus, the atomic force microscope (AFM)
      • Hahn C.A.
      • Zhou S.M.
      • Raynor R.
      • Tisch A.
      • Light K.
      • Shafman T.
      • Wong T.
      • Kirkpatrick J.
      • Turkington T.
      • Hollis D.
      • Marks L.B.
      Dose-dependent effects of radiation therapy on cerebral blood flow, metabolism, and neurocognitive dysfunction.
      has been shown to be a valuable tool for the quantification of the eGC nanomechanics on living endothelial cells. Since the eGC was first described as tethers at the surface of endothelial cells by Sun et al
      • Sun M.Z.
      • Graham J.S.
      • Hegedus B.
      • Marga F.
      • Zhang Y.
      • Forgacs G.
      • Grandbois M.
      Multiple membrane tethers probed by atomic force microscopy.
      >10 years ago, a number of relevant papers using the AFM technique for characterizing the nanomechanical properties of the eGC have been published.
      • Peters W.
      • Drueppel V.
      • Kusche-Vihrog K.
      • Schubert C.
      • Oberleithner H.
      Nanomechanics and sodium permeability of endothelial surface layer modulated by hawthorn extract WS 1442.
      ,
      • Sokolov I.
      • Dokukin M.E.
      • Guz N.V.
      Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments.
      • O'Callaghan R.
      • Job K.M.
      • Dull R.O.
      • Hlady V.
      Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy.
      • Marcotti S.
      • Maki K.
      • Reilly G.C.
      • Lacroix D.
      • Adachi T.
      Hyaluronic acid selective anchoring to the cytoskeleton: an atomic force microscopy study.
      • Kabedev A.
      • Lobaskin V.
      Structure and elasticity of bush and brush-like models of the endothelial glycocalyx.
      Using AFM as a nanoindentation tool is one of the most versatile techniques allowing the determination of mechanical properties of soft surfaces of living cells in the nanometer scale. For nanoindentation measurements in general, a spherical AFM tip, mounted to the end of a highly flexible cantilever, is used as a mechanical sensor as it is gradually lowered onto the cell and indents the cell membrane upon contacting the upper surface. The resulting deflection of the cantilever, which serves as a soft spring, is measured via reflection of a laser beam from the gold-coated backside of the cantilever and can be directly related to the stiffness of the cell. Cantilever deflection can thus be plotted as a function of tip position along the z axis. In order to quantify cell stiffness, the obtained data are transformed into a force-versus-deformation curve, using the cantilever's spring constant and the optical cantilever sensitivity. The slope of this force–distance curve then directly reflects the force (in nanonewtons, nN), here defined as stiffness, that has to be exerted to indent the cell for a certain distance.
      • Jeggle P.
      • Callies C.
      • Tarjus A.
      • Fassot C.
      • Fels J.
      • Oberleithner H.
      • Jaisser F.
      • Kusche-Vihrog K.
      Epithelial sodium channel stiffens the vascular endothelium in vitro and in Liddle mice.
      The AFM allows differentiation between different layers of the cell: i) eGC, ii) plasma membrane + submembranous cortex including the cortical cytoskeleton, and iii) the cell center.
      • Oberleithner H.
      • Peters W.
      • Kusche-Vihrog K.
      • Korte S.
      • Schillers H.
      • Kliche K.
      • Oberleithner K.
      Salt overload damages the glycocalyx sodium barrier of vascular endothelium.
      Technical requirements for probing the eGC with the AFM are the use of soft cantilevers, spherical tips with a diameter of approximately 10 μm, and a maximal loading force in the range of 0.5 to 5 nN. In Figure 2, a scheme of AFM-based nanoindentation measurements of the eGC is given. With this technique, nanomechanical properties of the eGC can be measured in vitro or ex vivo. For in vitro studies it is imperative that the cells have enough time to recover and assemble a fully developed eGC on their surface after the splitting procedure. In addition, an important prerequisite of proper endothelial function and fully pronounced endothelial characteristics, such as the expression of membrane receptors and ion channels, is a confluent monolayer of the endothelial cells. Normally, this takes at least 3 to 4 days under standard cell culture conditions.
      Figure thumbnail gr2
      Figure 2Model of different eGC conformations. Depending on different extracellular conditions, the eGC can exist in at least three different conformations: intact, collapse, or shed. The eGC conformation also depends on the polymerization state of the cortical cytoskeleton. A polymerized cortical actin favors a flat eGC, while actin depolymerization leads to a more upright eGC conformation. EC, endothelial cell.
      Some years ago, the AFM-based nanoindentation analysis of the eGC with vessels from mice and patients was established. Therefore, arteries are isolated and freed from surrounding tissue. Small patches (approximately 1 to 2 mm2) of the whole vessel are attached on glass, with the endothelial surface facing upward and allowed to recover with cell culture medium in the incubator for 24 to 48 hours. Afterward the height and stiffness can be assessed using the AFM.
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      With this method enhancement, the eGC can be studied in its native and physiological surroundings, and state-of-the-art mouse models and patient vessels can be included.

      Structure–Function Relationship of the Glycocalyx

      Importantly, under physiological conditions, the structure of the eGC layer is fairly stable but subject to a permanent dynamic balance between biosynthesis of new glycosaminoglycans and shear-dependent removal of existing constituents. Thus, the eGC must be a flexible and inhomogeneous structure due to various electrostatic and molecular interactions between its constituents.
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      ,
      • Mulivor A.W.
      • Lipowsky H.H.
      Inflammation- and ischemia-induced shedding of venular glycocalyx.
      Treatment with heparinase was shown to decrease the height and stiffness of the eGC (Figure 1, A and B).
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      Due to its position on top of endothelial cells, the eGC is subjected to various biophysical and biochemical factors from the streaming blood. Thus, under specific conditions that might be nonphysiological, the eGC can be shed, degraded, or collapsed, leading to measurable variations in height and stiffness of the structure. Importantly, such changes in nanomechanical properties are directly linked to functional changes of the cell surface. During the inflammatory process, the eGC is offended by proinflammatory cytokines such as tumor necrosis factor (TNF)-α or IL-1β, leading to the adhesion of leukocytes. As described above (Structure and Composition of the eGC), the eGC can reach a height of about 0.5 to 1 μm. Thus, attracted leukocytes are tip-toeing with their cytoskeletal protrusions on the eGC and can barely reach the adhesion molecules at the endothelial surface—unless the barrier is compromised. Recently, we showed that enzymatic removal of the eGC enhances adhesion forces between monocytes and the endothelial surface in a vascular cell adhesion molecule–dependent manner.
      • Schierke F.
      • Wyrwoll M.J.
      • Wisdorf M.
      • Niedzielski L.
      • Maase M.
      • Ruck T.
      • Meuth S.G.
      • Kusche-Vihrog K.
      Nanomechanics of the endothelial glycocalyx contribute to Na+-induced vascular inflammation.
      These findings indicate that a healthy eGC prevents the formation of bonds between binding partners on monocytes and endothelial surfaces. In addition, it has been shown that monocyte adhesion is facilitated after long-term treatment of vascular endothelial cells with high Na+.
      • Schierke F.
      • Wyrwoll M.J.
      • Wisdorf M.
      • Niedzielski L.
      • Maase M.
      • Ruck T.
      • Meuth S.G.
      • Kusche-Vihrog K.
      Nanomechanics of the endothelial glycocalyx contribute to Na+-induced vascular inflammation.
      The underlying explanation appears to be Na+-induced activation of endothelial cells, leading to endothelial-born IL-1β and TNF-α secretion and after conformational changes and damage of the eGC.
      • Schierke F.
      • Wyrwoll M.J.
      • Wisdorf M.
      • Niedzielski L.
      • Maase M.
      • Ruck T.
      • Meuth S.G.
      • Kusche-Vihrog K.
      Nanomechanics of the endothelial glycocalyx contribute to Na+-induced vascular inflammation.
      ,
      • Hijmans R.S.
      • Shrestha P.
      • Sarpong K.A.
      • Yazdani S.
      • El Masri R.
      • de Jong W.H.A.
      • Navis G.
      • Vives R.R.
      • van den Born J.
      High sodium diet converts renal proteoglycans into pro-inflammatory mediators in rats.
      As shown in Figure 1, C and D, treatment of endothelial cells with TNF-α leads to decreases in eGC height (flat eGC) and stiffness (soft eGC).
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      In this context, Szczygiel et al
      • Szczygiel A.M.
      • Brzezinka G.
      • Targosz-Korecka M.
      • Chlopicki S.
      • Szymonski M.
      Elasticity changes anti-correlate with NO production for human endothelial cells stimulated with TNF-alpha.
      reported that incubation of endothelial cells with TNF-α also affects the cortical stiffness due to actin polymerization. These processes were paralleled by reduced nitric oxide (NO) release, indicating beginning endothelial dysfunction under proinflammatory conditions. The fact that TNF-α manipulates the nanomechanical properties of both the eGC and cortex led to the assumption that both layers are strongly connected and mechanically interdependent.
      • Weinbaum S.
      • Zhang X.
      • Han Y.
      • Vink H.
      • Cowin S.C.
      Mechanotransduction and flow across the endothelial glycocalyx.
      Due to its position at the very surface of endothelial cells, the eGC is in direct contact with the plasma and thus subjected to the shear forces of the streaming blood. However, it is known that the eGC acts as a mechanosensor while shear stress in addition is a decisive factor for structural and functional changes of the eGC.
      • Weinbaum S.
      • Tarbell J.M.
      • Damiano E.R.
      The structure and function of the endothelial glycocalyx layer.
      At first glance, this statement seems to be contradictory, but the fact that the eGC combines these two functions, namely being both a transmitter and a receiver of the same signal, might explain specific regulatory processes concerning the endothelial surface, including feedback reinforcement in endothelial function.
      • Kroger N.
      • Brand R.
      • van Biezen A.
      • Zander A.
      • Dierlamm J.
      • Niederwieser D.
      • Devergie A.
      • Ruutu T.
      • Cornish J.
      • Ljungman P.
      • Gratwohl A.
      • Cordonnier C.
      • Beelen D.
      • Deconinck E.
      • Symeonidis A.
      • de Witte T.
      Myelodysplastic Syndromes Subcommittee of The Chronic Leukaemia Working Party of European Group for Blood and Marrow Transplantation
      Risk factors for therapy-related myelodysplastic syndrome and acute myeloid leukemia treated with allogeneic stem cell transplantation.
      From a pathophysiological point of view, a disturbed mechanotransduction of the damaged eGC to the endothelial cortex will lead to changes in membrane characteristics, including the presence of anchor proteins, membrane-bound ion channels, and receptors, and thus in turn to impaired nanomechanical properties and function of the eGC (in other words, a vicious circle).
      • Zhang X.
      • Sun D.
      • Song J.W.
      • Zullo J.
      • Lipphardt M.
      • Coneh-Gould L.
      • Goligorsky M.S.
      Endothelial cell dysfunction and glycocalyx - a vicious circle.
      A consequence of the disturbed mechanosensing caused by a dysfunctional eGC is the perturbation of intracellular signaling events, including, among others, cytosolic Ca2+ and endothelial NO synthase
      • Dragovich M.A.
      • Chester D.
      • Fu B.M.
      • Wu C.
      • Xu Y.
      • Goligorsky M.S.
      • Zhang X.F.
      Mechanotransduction of the endothelial glycocalyx mediates nitric oxide production through activation of TRP channels.
      ; ρ family GTPases and tyrosine kinases Ras, ERK, and c-Jun N-terminal kinase
      • Takahashi M.
      • Ishida T.
      • Traub O.
      • Corson M.A.
      • Berk B.C.
      Mechanotransduction in endothelial cells: temporal signaling events in response to shear stress.
      ; and cytoskeletal remodeling.
      • Ando J.
      • Yamamoto K.
      Flow detection and calcium signalling in vascular endothelial cells.
      The eGC is stabilized by shear stress, which in turn is pivotal for proper NO production.
      • Thi M.M.
      • Tarbell J.M.
      • Weinbaum S.
      • Spray D.C.
      The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: a "bumper-car" model.
      ,
      • Koo A.
      • Dewey Jr., C.F.
      • Garcia-Cardena G.
      Hemodynamic shear stress characteristic of atherosclerosis-resistant regions promotes glycocalyx formation in cultured endothelial cells.
      In this context, it was demonstrated that heparan sulfate proteoglycans are crucial mechanosensors mediating regulatory NO release.
      • Florian J.A.
      • Kosky J.R.
      • Ainslie K.
      • Pang Z.
      • Dull R.O.
      • Tarbell J.M.
      Heparan sulfate proteoglycan is a mechanosensor on endothelial cells.
      When the eGC is shed after heparinase treatment, this effect can be completely abrogated, emphasizing the important role of the eGC under fluid shear stress. Furthermore, heparanase-dependent eGC breakdown has been shown to contribute to plasma leakage and leukocyte recruitment in vivo
      • Lukasz A.
      • Hillgruber C.
      • Oberleithner H.
      • Kusche-Vihrog K.
      • Pavenstadt H.
      • Rovas A.
      • Hesse B.
      • Goerge T.
      • Kumpers P.
      Endothelial glycocalyx breakdown is mediated by angiopoietin-2.
      —further supporting the need for an intact eGC under flow conditions, providing a wide range of vasculoprotective properties.
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      ,
      • Lukasz A.
      • Hillgruber C.
      • Oberleithner H.
      • Kusche-Vihrog K.
      • Pavenstadt H.
      • Rovas A.
      • Hesse B.
      • Goerge T.
      • Kumpers P.
      Endothelial glycocalyx breakdown is mediated by angiopoietin-2.
      ,
      • Gouverneur M.
      • Berg B.
      • Nieuwdorp M.
      • Stroes E.
      • Vink H.
      Vasculoprotective properties of the endothelial glycocalyx: effects of fluid shear stress.
      Yet, there are also reports suggesting that shear stress per se could lead to shedding and damage the mechanosensitive properties of the eGC.
      • Zeng Y.
      • Tarbell J.M.
      The adaptive remodeling of endothelial glycocalyx in response to fluid shear stress.
      However, the stabilization and building-up of the eGC by shear stress might rather be a physiological response to its shear stress sensing and its mechanotransductory function under flow conditions. In agreement with recent findings by Zeng and Tarbell,
      • Zeng Y.
      • Tarbell J.M.
      The adaptive remodeling of endothelial glycocalyx in response to fluid shear stress.
      we have shown that the application of moderate laminar shear stress (8 dyne/cm2) increased the amount of HS at the surface of endothelial cells, while treatment of the cells with heparinase I led to a significant reduction and compromising of the eGC under shear stress conditions (Z.C.C. and K.K.V., unpublished data). These findings indicate that physiological shear stress is imperative for proper structure and integrity of the eGC and thus for the functional demands of the vascular system.
      The cortical cytoskeleton was considered a highly dynamic structure,
      • Fels J.
      • Jeggle P.
      • Kusche-Vihrog K.
      • Oberleithner H.
      Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.
      and it is known that fluid shear stress induces cytoskeletal reorganization.
      • Cucina A.
      • Sterpetti A.V.
      • Pupelis G.
      • Fragale A.
      • Lepidi S.
      • Cavallaro A.
      • Giustiniani Q.
      • D'Angelo L.S.
      Shear stress induces changes in the morphology and cytoskeleton organisation of arterial endothelial cells.
      Additionally, the cell cortex seems to influence endothelium-specific mechanisms and signaling cascades involved in cell cycle, migration, and aging.
      • Stewart M.P.
      • Helenius J.
      • Toyoda Y.
      • Ramanathan S.P.
      • Muller D.J.
      • Hyman A.A.
      Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding.
      • Rotsch C.
      • Jacobson K.
      • Radmacher M.
      Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy.
      • Laurent V.M.
      • Kasas S.
      • Yersin A.
      • Schaffer T.E.
      • Catsicas S.
      • Dietler G.
      • Verkhovsky A.B.
      • Meister J.J.
      Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy.
      • Sokolov I.
      • Iyer S.
      • Woodworth C.D.
      Recovery of elasticity of aged human epithelial cells in vitro.
      Reorganization of the cortical actin cytoskeleton has been shown to significantly contribute to changes in endothelial nanomechanics and function
      • Kronlage C.
      • Schafer-Herte M.
      • Boning D.
      • Oberleithner H.
      • Fels J.
      Feeling for filaments: quantification of the cortical actin web in live vascular endothelium.
      ,
      • Prystopiuk V.
      • Fels B.
      • Simon C.S.
      • Liashkovich I.
      • Pasrednik D.
      • Kronlage C.
      • Wedlich-Soldner R.
      • Oberleithner H.
      • Fels J.
      A two-phase response of endothelial cells to hydrostatic pressure.
      —which might ultimately result in an impaired production of the vasoactive regulator NO. These findings are supported by studies showing that endothelial NO synthase activity is increased by globular actin
      • Kondrikov D.
      • Han H.R.
      • Block E.R.
      • Su Y.
      Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells.
      ,
      • Su Y.
      • Edwards-Bennett S.
      • Bubb M.R.
      • Block E.R.
      Regulation of endothelial nitric oxide synthase by the actin cytoskeleton.
      —a property of a soft endothelial cell exhibiting a larger globular actin/filamentous (F)-actin ratio—and thus possesses a better capacity to release NO during shear stress.
      • Fels J.
      • Callies C.
      • Kusche-Vihrog K.
      • Oberleithner H.
      Nitric oxide release follows endothelial nanomechanics and not vice versa.
      That means that a proper cell deformability by fluid shear stress is pivotal for NO release and maintenance of a physiological endothelial function. In agreement with recently published data,
      • Zeng Y.
      • Tarbell J.M.
      The adaptive remodeling of endothelial glycocalyx in response to fluid shear stress.
      we have shown that moderate laminar shear stress (8 dyne/cm2) significantly increased the amount of cortical F-actin, meaning that the filamentous form of actin was stabilized by shear stress and most likely favored compared to its globular form (Z.C.C. and K.K.V., unpublished data). This finding indicates that acute laminar shear stress leads to remodeling and stiffening of the cortical cytoskeleton. Thus, shear stress seems to affect different layers of the outer shell of endothelial cells. The working model is that changes in blood flow affect the conformation of the eGC, which transmits the primary mechanical signal to the cytoskeleton through the intracellular domains of the eGC. Generally, flow detection seems to work through a direct connection between the eGC and cytoskeletal proteins in the cytosol and cell cortex.
      • Ando J.
      • Yamamoto K.
      Flow detection and calcium signalling in vascular endothelial cells.
      In addition to this sole mechanical view, the transduction of the signals from shear stress into biochemical signals via changes in local concentration gradients of ions must be considered. Recently, our group studied the effects of high physiological plasma Na+ on the nanomechanical properties of eGC and cortex. We found that a plasma Na+ concentration of >145 mmol/L: i) deteriorates the eGC (Table 1), ii) stiffens the endothelial cortex, and iii) polymerizes the cortical actin.
      • Oberleithner H.
      • Peters W.
      • Kusche-Vihrog K.
      • Korte S.
      • Schillers H.
      • Kliche K.
      • Oberleithner K.
      Salt overload damages the glycocalyx sodium barrier of vascular endothelium.
      ,
      • Schierke F.
      • Wyrwoll M.J.
      • Wisdorf M.
      • Niedzielski L.
      • Maase M.
      • Ruck T.
      • Meuth S.G.
      • Kusche-Vihrog K.
      Nanomechanics of the endothelial glycocalyx contribute to Na+-induced vascular inflammation.
      ,
      • Korte S.
      • Strater A.S.
      • Druppel V.
      • Oberleithner H.
      • Jeggle P.
      • Grossmann C.
      • Fobker M.
      • Nofer J.R.
      • Brand E.
      • Kusche-Vihrog K.
      Feedforward activation of endothelial ENaC by high sodium.
      Interestingly, an acute increase in potassium in the physiological range softens the endothelial cortex and increases the release of NO.
      • Oberleithner H.
      • Callies C.
      • Kusche-Vihrog K.
      • Schillers H.
      • Shahin V.
      • Riethmuller C.
      • MacGregor G.A.
      • de Wardener H.E.
      Potassium softens vascular endothelium and increases nitric oxide release.
      From these data it can be concluded that the damage of the eGC enables the Na+ influx into the cell via endothelial sodium channels (ENaC). In the interior of the cell, the local increase in Na+ leads to conformational changes in cortical actin (shift from globular actin to F-actin), which in turn mechanically stiffens the cortex. As a consequence, the release of NO is reduced and the adhesion of leukocytes is facilitated, indicating the development of vascular inflammation.
      • Schierke F.
      • Wyrwoll M.J.
      • Wisdorf M.
      • Niedzielski L.
      • Maase M.
      • Ruck T.
      • Meuth S.G.
      • Kusche-Vihrog K.
      Nanomechanics of the endothelial glycocalyx contribute to Na+-induced vascular inflammation.
      ,
      • Oberleithner H.
      • Riethmuller C.
      • Schillers H.
      • MacGregor G.A.
      • de Wardener H.E.
      • Hausberg M.
      Plasma sodium stiffens vascular endothelium and reduces nitric oxide release.
      Table 1Changes in the Nanomechanical Properties of the eGC
      PropertyeGC height, % compared with control conditionseGC stiffness, % compared with control conditions
      High sodium
      • Tkachenko E.
      • Rhodes J.M.
      • Simons M.
      Syndecans: new kids on the signaling block.
      −50+130
      TNFα
      • Prystopiuk V.
      • Fels B.
      • Simon C.S.
      • Liashkovich I.
      • Pasrednik D.
      • Kronlage C.
      • Wedlich-Soldner R.
      • Oberleithner H.
      • Fels J.
      A two-phase response of endothelial cells to hydrostatic pressure.
      −55−35
      Heparinase
      • Prystopiuk V.
      • Fels B.
      • Simon C.S.
      • Liashkovich I.
      • Pasrednik D.
      • Kronlage C.
      • Wedlich-Soldner R.
      • Oberleithner H.
      • Fels J.
      A two-phase response of endothelial cells to hydrostatic pressure.
      −502−33
      Stiff cortex
      • Kroger N.
      • Brand R.
      • van Biezen A.
      • Zander A.
      • Dierlamm J.
      • Niederwieser D.
      • Devergie A.
      • Ruutu T.
      • Cornish J.
      • Ljungman P.
      • Gratwohl A.
      • Cordonnier C.
      • Beelen D.
      • Deconinck E.
      • Symeonidis A.
      • de Witte T.
      Myelodysplastic Syndromes Subcommittee of The Chronic Leukaemia Working Party of European Group for Blood and Marrow Transplantation
      Risk factors for therapy-related myelodysplastic syndrome and acute myeloid leukemia treated with allogeneic stem cell transplantation.
      −8+9
      Soft cortex
      • Kroger N.
      • Brand R.
      • van Biezen A.
      • Zander A.
      • Dierlamm J.
      • Niederwieser D.
      • Devergie A.
      • Ruutu T.
      • Cornish J.
      • Ljungman P.
      • Gratwohl A.
      • Cordonnier C.
      • Beelen D.
      • Deconinck E.
      • Symeonidis A.
      • de Witte T.
      Myelodysplastic Syndromes Subcommittee of The Chronic Leukaemia Working Party of European Group for Blood and Marrow Transplantation
      Risk factors for therapy-related myelodysplastic syndrome and acute myeloid leukemia treated with allogeneic stem cell transplantation.
      +13−16
      As described above (Structure and Composition of the eGC), the eGC can be changed in two parameters: height/thickness and stiffness. The combination of both factors might be indicative of the underlying mechanism of the process.
      Proinflammatory cytokines such as TNF-α lead to a flat and soft eGC (Table 1)
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      and can thus be interpreted as a process of shedding. This means that some of the eGC components are simply removed, resulting in a thinned eGC. The same can be observed by the application of classic sheddases such as heparinase (Figure 1, A and B, and Table 1).
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      In contrast, when plasma Na+ concentrations exceed a specific threshold, the stiffness of the eGC is increased, while its functional thickness is reduced. Such structural changes are rather indicative of a collapse of the structure. This condition occurs most likely via a Na+-dependent reduction in the negatively charged HS residues of the eGC.
      • Oberleithner H.
      • Peters W.
      • Kusche-Vihrog K.
      • Korte S.
      • Schillers H.
      • Kliche K.
      • Oberleithner K.
      Salt overload damages the glycocalyx sodium barrier of vascular endothelium.
      ,
      • Bevan J.A.
      Flow regulation of vascular tone. Its sensitivity to changes in sodium and calcium.
      ,
      • Siegel G.
      • Walter A.
      • Kauschmann A.
      • Malmsten M.
      • Buddecke E.
      Anionic biopolymers as blood flow sensors.
      In this context, it can be speculated that changes in the eGC height depend solely on the amount of components building up the surface layer, which is relevant in inflammation-induced eGC damage. The functional causality of changes in eGC stiffness seem to be more complex as they reflect a combination of charge-dependent collapse and deterioration of eGC components.
      In this context, it is worth mentioning that the eGC provides a repository for a variety of biologically active molecules, as it incorporates and interacts with extracellular superoxide dismutase; xanthine oxidoreductase; interleukins such as IL-2 to -5, -7, -8, and -12; low-density lipoprotein
      • Wildling L.
      • Hinterdorfer P.
      • Kusche-Vihrog K.
      • Treffner Y.
      • Oberleithner H.
      Aldosterone receptor sites on plasma membrane of human vascular endothelium detected by a mechanical nanosensor.
      and low-density lipoprotein lipase, basic fibroblast growth factor, vascular endothelial growth factor, and TGF-β; and several regulators of coagulation, such as antithrombin III, heparin cofactor II, and tissue pathway factor inhibitor.
      • Reitsma S.
      • Slaaf D.W.
      • Vink H.
      • van Zandvoort M.A.
      • Oude Egbrink M.G.
      The endothelial glycocalyx: composition, functions, and visualization.
      ,
      • Zhang X.
      • Sun D.
      • Song J.W.
      • Zullo J.
      • Lipphardt M.
      • Coneh-Gould L.
      • Goligorsky M.S.
      Endothelial cell dysfunction and glycocalyx - a vicious circle.
      In case of eGC degradation, these molecules might be released and the process would become self-perpetuating.
      From a functional point of view, all possible modifications of the nanomechanical properties of the endothelial surface are important in the context of its barrier function. Erythrocytes, for example, must be prevented from touching the endothelial wall, and a frictionless slipping through the narrow vessel must be guaranteed. Therefore, an upright and negatively charged eGC is needed. In contrast, leukocytes must breach the endothelial barrier, including the eGC, to extravasate across the vascular endothelium.
      • Celie J.W.
      • Beelen R.H.
      • van den Born J.
      Heparan sulfate proteoglycans in extravasation: assisting leukocyte guidance.
      Here, the individual steps of leukocyte transmigration are mediated by adhesion receptors and an appropriate stiffness of the endothelium.
      • Schaefer A.
      • Hordijk P.L.
      Cell-stiffness-induced mechanosignaling - a key driver of leukocyte transendothelial migration.
      It is postulated that the eGC has both pro- and antiadhesive functions, and thus its nanomechanical properties play a crucial role during leukocyte recruitment and inflammatory processes.
      The eGC can exist in three different conformations that fulfill individual physiological roles: i) intact (upright and soft), ii) collapsed (flat and stiff), and iii) shed (flat and soft) (Figure 1). A soft and expanded eGC is supposed to indicate a fully functional endothelium, whereas a shed or collapsed eGC most likely exerts adverse effects on the vascular system and has been discussed as being a strong promoter of vascular diseases.
      • Fels J.
      • Kusche-Vihrog K.
      Endothelial nanomechanics in the context of endothelial (dys)function and inflammation.

      Mutual Interference between Glycocalyx and Endothelial Cortex

      The endothelial cortex, an actin-rich layer 50 to 150 nm beneath the plasma membrane, represents a vulnerable regulatory compartment that is able to rapidly change its mechanical properties to react to functional challenges and physiological adaptations. Together with the eGC, the cortical region senses and processes the mechanical stress from the streaming blood, resulting in the release or repression of vasoactive substances such as NO. Using the AFM as a nanosensor, we have shown that mechanical stiffening of the endothelial cortex reduces the release of NO.
      • Fels J.
      • Callies C.
      • Kusche-Vihrog K.
      • Oberleithner H.
      Nitric oxide release follows endothelial nanomechanics and not vice versa.
      The cortex mainly consists of F-actin, the main network constituent and the motor protein myosin II, which functions as a point force generator and actin crosslinker.
      • Ayala Y.A.
      • Pontes B.
      • Hissa B.
      • Monteiro A.C.
      • Farina M.
      • Moura-Neto V.
      • Viana N.B.
      • Nussenzveig H.M.
      Effects of cytoskeletal drugs on actin cortex elasticity.
      Cortical stiffening was brought in context with a shift from globular actin to F-actin, resulting in an increase in the amount of cortical actin fibers.
      • Fels J.
      • Jeggle P.
      • Kusche-Vihrog K.
      • Oberleithner H.
      Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.
      Compared to a soft cortex, a stiff cortex cannot be deformed easily by the streaming blood resulting in a reduced stimulus for endothelial NO synthase activation and thus NO release. This inverse correlation between cortical stiffness and NO secretion is physiologically relevant since it locally can regulate the vasotonus of the vessels and thus counteract blood pressure variations immediately.
      • Warnock D.G.
      • Kusche-Vihrog K.
      • Tarjus A.
      • Sheng S.
      • Oberleithner H.
      • Kleyman T.R.
      • Jaisser F.
      Blood pressure, and amiloride-sensitive sodium channels in vascular and renal cells.
      From a pathophysiological view, a sustained stiffening of the endothelial surface causes impaired vasodilation of blood vessels. Recently, this was described as the stiff endothelial cell syndrome.
      • Lang F.
      Stiff endothelial cell syndrome in vascular inflammation and mineralocorticoid excess.
      A flexible switch between stiff and soft endothelial cortex is necessary under normal conditions to enable proper vascular function. This endothelial plasticity is based on lively mechanical and biochemical interaction between the different layers. If this endothelial plasticity is lost, the development of endothelial dysfunction, arterial hypertension, and inflammatory processes might be in progress.
      • Liao J.K.
      Linking endothelial dysfunction with endothelial cell activation.
      ,
      • Lenders M.
      • Hofschroer V.
      • Schmitz B.
      • Kasprzak B.
      • Rohlmann A.
      • Missler M.
      • Pavenstadt H.
      • Oberleithner H.
      • Brand S.M.
      • Kusche-Vihrog K.
      • Brand E.
      Differential response to endothelial epithelial sodium channel inhibition ex vivo correlates with arterial stiffness in humans.
      Thus, the mechanical stiffness of the cortex can be seen as hallmark of endothelial dysfunction.
      The mechanical properties of the cortex can be manipulated by using agents interfering with actin polymerization, such as cytochalasin (Cy)-D and jasplakinolide. CyD stops actin polymerization by interrupting the elongation of actin filaments. The disruption of the F-actin network reduces filament length and alters the network properties. In contrast, jasplakinolide inhibits the turnover of actin filaments as well as descension of the newly synthesized filaments. By using optical tweezers, Ayala et al
      • Ayala Y.A.
      • Pontes B.
      • Hissa B.
      • Monteiro A.C.
      • Farina M.
      • Moura-Neto V.
      • Viana N.B.
      • Nussenzveig H.M.
      Effects of cytoskeletal drugs on actin cortex elasticity.
      found that both the elastic and viscous moduli decrease with CyD application as a consequence of the network disruption, indicating that CyD is a potent actin network disorganizer. Employing fast-scanning AFM, it was shown that cytochalasin B, which inhibits actin monomers from being added to the barbed end of filaments, significantly reduced the rate of polymerization, while the addition of Jas inhibited not only polymerization/depolymerization turnover of cortical actin but also the descending movement of the filaments.
      • Zhang Y.
      • Yoshida A.
      • Sakai N.
      • Uekusa Y.
      • Kumeta M.
      • Yoshimura S.H.
      In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy.
      As described above (Structure and Composition of the eGC), the eGC is directly connected to cytoskeletal proteins with its cytosolic parts, leading to the assumption that both layers are mechanically and functionally connected. In Figure 3, data are summarized that give a strong hint on the interdependency of eGC and cortex.
      • Peters W.
      • Kusche-Vihrog K.
      • Oberleithner H.
      • Schillers H.
      Cystic fibrosis transmembrane conductance regulator is involved in polyphenol-induced swelling of the endothelial glycocalyx.
      ,
      • Grimm K.B.
      • Oberleithner H.
      • Fels J.
      Fixed endothelial cells exhibit physiologically relevant nanomechanics of the cortical actin web.
      In agreement with published data, manipulation of the cortical actin results in differences in the cortical stiffness quantified with nanonindentation approaches using the AFM.
      • Peters W.
      • Kusche-Vihrog K.
      • Oberleithner H.
      • Schillers H.
      Cystic fibrosis transmembrane conductance regulator is involved in polyphenol-induced swelling of the endothelial glycocalyx.
      Application of the depolymerization agent CyD (50 nmol/L) decreases the cortical stiffness of endothelial cells within minutes (Figure 3A). In parallel, underlining the mechanical measurements, CyD reduces the amount of F-actin in the cortical cytoskeleton, indicating the shift mentioned in the previous paragraph from filamentous to globular actin (Figure 3B). Since specific components of the eGC are anchored to the actin cytoskeleton,
      • Marcotti S.
      • Maki K.
      • Reilly G.C.
      • Lacroix D.
      • Adachi T.
      Hyaluronic acid selective anchoring to the cytoskeleton: an atomic force microscopy study.
      ,
      • Ando J.
      • Yamamoto K.
      Flow detection and calcium signalling in vascular endothelial cells.
      we followed the idea that manipulation of the stiffness degree of the endothelial cortex might change the nanomechanical properties of the eGC. As shown in Figure 3, C and D, this is the case: Depolymerization of the cortical actin with CyD reduced the stiffness of the eGC, while the height was increased, leading to a soft and upright eGC structure compared to control conditions.
      • Peters W.
      • Kusche-Vihrog K.
      • Oberleithner H.
      • Schillers H.
      Cystic fibrosis transmembrane conductance regulator is involved in polyphenol-induced swelling of the endothelial glycocalyx.
      Interestingly, these findings are in agreement with the effects of high extracellular plasma Na+ conditions. As mentioned in the previous paragraph, a high-Na+ environment stiffens the cortex of endothelial cells while the eGC becomes stiff and flat. Since Na+ overload leads—just as Jasplakinolide —to a polymerization of the cortical actin, a similar mechanism behind the cortex-dependent eGC collapse can be postulated. From these data it can be further speculated that the observed eGC collapse is due to an intrinsic signal coming from the cortical region of the cell, in terms that losing the anchoring of the eGC to components of the cortex destabilizes the complete outer layer of endothelial cells. In this regard, it should be kept in mind that also ion channels are connected with their cytosolic parts to the actin meshwork beneath the plasma membrane (eg, ENaC
      • Mazzochi C.
      • Benos D.J.
      • Smith P.R.
      Interaction of epithelial ion channels with the actin-based cytoskeleton.
      ). Thus, changes in the conductance of ion channels may manipulate the polymerization level of the actomyosin network and in turn the nanomechanical properties of the eGC. In summary, these facts and the following considerations strongly indicate that the eGC on top of cells, together with the proteins of the cortical cytoskeleton, build a highly dynamic regulatory hub that is both a transmitter and a target of mechanical signals from the direct environment. This mechanism enables the cells to immediately react to various conditions and to accordingly adjust their nanomechanical properties.
      Figure thumbnail gr3
      Figure 3Interdependency of the nanomechanical properties of eGC and cortex. Nanoindentation analysis probing both eGC and cortex revealed that the application of cytochalasin D (CyD) (50 nmol/L) reduces the cortical stiffness compared to control conditions (A), the amount of cortical F-actin (B), and the stiffness of the eGC (C), while its height is increased (D). Data are expressed as means ± SEM. n = 3 to 5. ∗P < 0.05. Data in A modified from Peters et al
      • Peters W.
      • Kusche-Vihrog K.
      • Oberleithner H.
      • Schillers H.
      Cystic fibrosis transmembrane conductance regulator is involved in polyphenol-induced swelling of the endothelial glycocalyx.
      and Grimm et al
      • Grimm K.B.
      • Oberleithner H.
      • Fels J.
      Fixed endothelial cells exhibit physiologically relevant nanomechanics of the cortical actin web.
      ; data in B modified from Grimm et al
      • Grimm K.B.
      • Oberleithner H.
      • Fels J.
      Fixed endothelial cells exhibit physiologically relevant nanomechanics of the cortical actin web.
      ; data in D modified from Peters et al.
      • Peters W.
      • Kusche-Vihrog K.
      • Oberleithner H.
      • Schillers H.
      Cystic fibrosis transmembrane conductance regulator is involved in polyphenol-induced swelling of the endothelial glycocalyx.

      Nanomechanics of the Glycocalyx and Clinical Relevance

      Many diseases are accompanied by a deterioration of the eGC. Specifically, inflammatory conditions may cause massive damage of the eGC, resulting in enhanced vascular permeability, tissue edema, augmented leukocyte adhesion, platelet aggregation, and dysregulated vasodilation.
      • Tarbell J.M.
      • Cancel L.M.
      The glycocalyx and its significance in human medicine.
      Thus, the innovative structural flexibility of the eGC is at the expense of its resistance and resilience during nonphysiological situations, such as illness or surgery. In septic patients, it was found that eGC components (eg, syndecan-1, heparan sulfate, hyaluronan, chondroitin sulfates) are released into the plasma and may serve as clinically relevant biomarkers.
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      Several enzymes mediate this degradation. Heparanase directly cleaves the heparan sulfate chains attached to core proteoglycans. Metalloproteinases are known to cleave proteoglycans (eg, syndecan-1) directly from the endothelial cell membrane. These specific enzymes are activated in the inflammatory state by reactive oxygen species and proinflammatory cytokines such as TNF-α and IL-1β. Elevated heparanase expression can secondarily increase metalloproteinase expression in myeloma cells, suggesting crosstalk between sheddases.
      • Schmidt E.P.
      • Yang Y.
      • Janssen W.J.
      • Gandjeva A.
      • Perez M.J.
      • Barthel L.
      • Zemans R.L.
      • Bowman J.C.
      • Koyanagi D.E.
      • Yunt Z.X.
      • Smith L.P.
      • Cheng S.S.
      • Overdier K.H.
      • Thompson K.R.
      • Geraci M.W.
      • Douglas I.S.
      • Pearse D.B.
      • Tuder R.M.
      The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.
      ,
      • Becker B.F.
      • Jacob M.
      • Leipert S.
      • Salmon A.H.
      • Chappell D.
      Degradation of the endothelial glycocalyx in clinical settings: searching for the sheddases.
      AFM-based studies revealed that inflammation-dependent deterioration processes are based on modifications of the eGC nanomechanics. As mentioned above (Structure and Composition of the eGC), the application of TNF-α to endothelial cells in vitro leads to a softer and flatter eGC, which might be due to shedding-related thinning of the structure (Table 1).
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      This shedding breaks the vasculoprotective barrier as such and additionally may lead to the release of proinflammatory cytokines stored in the eGC layer.
      • Zhang X.
      • Sun D.
      • Song J.W.
      • Zullo J.
      • Lipphardt M.
      • Coneh-Gould L.
      • Goligorsky M.S.
      Endothelial cell dysfunction and glycocalyx - a vicious circle.
      Wiesinger et al
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      took advantage of the sophisticated AFM-based principle to exactly quantify the nanomechanical properties of the eGC in a preclinical approach. They found that ex vivo endothelial cells derived from mouse aorta in situ preparations treated in vivo with lipopolysaccharide had a significantly reduced eGC height and stiffness, indicating severe damage. In addition, acute application of heparinase I, thrombin, lipopolysaccharide, or TNF-α alone to microvascular endothelial cells in vitro was sufficient to sustainably damage the eGC.
      • Wiesinger A.
      • Peters W.
      • Chappell D.
      • Kentrup D.
      • Reuter S.
      • Pavenstadt H.
      • Oberleithner H.
      • Kumpers P.
      Nanomechanics of the endothelial glycocalyx in experimental sepsis.
      During the past few years, a method of quantification and visualization of the eGC in vivo with patients was developed by Vink and colleagues.
      • Van Teeffelen J.W.
      • Brands J.
      • Stroes E.S.
      • Vink H.
      Endothelial glycocalyx: sweet shield of blood vessels.
      With the so-called sublingual microcirculatory measurements using sidestream dark field imaging, the perfused boundary region in the microvessels of the tongue could be studied in patients. Thereby, the width of the perfused boundary region serves as an inverse parameter of eGC dimensions in vessels with diameters of between 5 and 25 μm in vivo.
      • Rovas A.
      • Lukasz A.H.
      • Vink H.
      • Urban M.
      • Sackarnd J.
      • Pavenstadt H.
      • Kumpers P.
      Bedside analysis of the sublingual microvascular glycocalyx in the emergency room and intensive care unit - the GlycoNurse study.
      Pilot studies conducted in the intensive care unit revealed that the perfused boundary region is indeed markedly increased in critically ill patients compared to healthy controls. Employing an automated acquisition of the data, this technique might be feasible and reproducible in the emergency department to identify patients at high risk for organ failure and death under routine conditions. Another elegant and noninvasive approach to determining eGC nanomechanics and integrity was developed by Oberleithner few years ago.
      • Oberleithner H.
      Sodium selective erythrocyte glycocalyx and salt sensitivity in man.
      ,
      • Oberleithner H.
      Vascular endothelium leaves fingerprints on the surface of erythrocytes.
      With the so-called salt blood test mini, a droplet of capillary blood is mixed with a smart Na+ cocktail. Then the red blood cells of this mixture are allowed to undergo sedimentation by gravity in a glass tube. In this setting, a fast sedimentation indicates disturbed eGC integrity and can be a useful clinical marker of endothelial dysfunction and predictor of the development of cardiovascular pathologies.
      • Oberleithner H.
      Sodium selective erythrocyte glycocalyx and salt sensitivity in man.
      ,
      • Oberleithner H.
      Vascular endothelium leaves fingerprints on the surface of erythrocytes.
      Damage of the eGC occurs not only during inflammation, infection, and sepsis but also during surgeries, especially when ischemia/reperfusion injury occurs. Because of a massive increase in oxidative stress triggered by ischemia/reperfusion injury, the eGC becomes dysfunctional. A postoperative increase in syndecan-1 level, for example, is correlated with subsequent complications.
      • Holzmann M.S.
      • Winkler M.S.
      • Strunden M.S.
      • Izbicki J.R.
      • Schoen G.
      • Greiwe G.
      • Pinnschmidt H.O.
      • Poppe A.
      • Saugel B.
      • Daum G.
      • Goetz A.E.
      • Heckel K.
      Syndecan-1 as a biomarker for sepsis survival after major abdominal surgery.
      In this case, either a rapid restoration or preoperative stabilization would be of great benefit to the patient. Efforts have been made to find a solution for this clinical problem. A variety of pharmacologic agents have been tested in vitro to restore or prevent the perioperative degradation of the eGC. Promising results have been obtained from, for example, antithrombin III, metalloproteinase inhibitors, or NO donors.
      • Song J.W.
      • Zullo J.
      • Lipphardt M.
      • Dragovich M.
      • Zhang F.X.
      • Fu B.
      • Goligorsky M.S.
      Endothelial glycocalyx-the battleground for complications of sepsis and kidney injury.
      The recent invention of liposomal nanocarriers of preassembled eGC showed that they are able to restore the eGC of endothelial cells in vitro.
      • Zhang X.
      • Sun D.
      • Song J.W.
      • Zullo J.
      • Lipphardt M.
      • Coneh-Gould L.
      • Goligorsky M.S.
      Endothelial cell dysfunction and glycocalyx - a vicious circle.
      Whether these liposomes exert a remedial effect on the nanomechanical properties of demonstrably damaged eGC in vivo remains to be studied.
      Unpublished observations from our laboratory (Z.C.C.) have shown that uremic serum derived from dialysis patients with chronic kidney disease drastically change the nanomechanical properties of the endothelial surface when added for 24 hours to the cell culture medium instead of fetal calf serum. It stiffens the cell cortex and deteriorates the eGC in a chronic kidney disease stage–dependent manner. Among others, this finding gives cause to optimism that studying the nanomechanical properties of the eGC under both physiological and pathophysiological conditions one day might lead to a precise and easy-to-handle diagnostic tool.
      Many years ago, Pitt et al
      • Pitt B.
      • Zannad F.
      • Remme W.J.
      • Cody R.
      • Castaigne A.
      • Perez A.
      • Palensky J.
      • Wittes J.
      The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators.
      demonstrated in the Randomized Aldactone Evaluation Study that the aldosterone receptor antagonist spironolactone, when added to therapy in heart failure patients, improved endothelial function, suggesting a role for aldosterone as part of the activated renin–angiotensin–aldosterone system in the endothelial dysfunction of heart failure. These results may partially explain the beneficial effects of mineralocorticoid antagonism in chronic heart failure in the Randomized Aldactone Evaluation Study, as well as in the recent Eplerenone Post-Acute Myocardial Infarction Heart failure Efficacy and Survival Study
      • Pitt B.
      • Williams G.
      • Remme W.
      • Martinez F.
      • Lopez-Sendon J.
      • Zannad F.
      • Neaton J.
      • Roniker B.
      • Hurley S.
      • Burns D.
      • Bittman R.
      • Kleiman J.
      The EPHESUS trial: eplerenone in patients with heart failure due to systolic dysfunction complicating acute myocardial infarction. Eplerenone Post-AMI Heart Failure Efficacy and Survival Study.
      of the more selective aldosterone receptor antagonist eplerenone in post–myocardial infarction subjects. Importantly, spironolactone and eplerenone are able to improve endothelial function by softening of the endothelial cortex.
      • Hillebrand U.
      • Schillers H.
      • Riethmüller C.
      • Stock C.
      • Wilhelmi M.
      • Oberleithner H.
      • Hausberg M.
      Dose-dependent endothelial cell growth and stiffening by aldosterone: endothelial protection by eplerenone.
      ,
      • Druppel V.
      • Kusche-Vihrog K.
      • Grossmann C.
      • Gekle M.
      • Kasprzak B.
      • Brand E.
      • Pavenstadt H.
      • Oberleithner H.
      • Kliche K.
      Long-term application of the aldosterone antagonist spironolactone prevents stiff endothelial cell syndrome.
      In addition, spironolactone may prevent Na+-induced eGC damage.
      • Oberleithner H.
      • Peters W.
      • Kusche-Vihrog K.
      • Korte S.
      • Schillers H.
      • Kliche K.
      • Oberleithner K.
      Salt overload damages the glycocalyx sodium barrier of vascular endothelium.
      Taking this into account, aldosterone antagonism should be considered as preventive and/or curative in patients with eGC-damaging situations. Thus, the eGC might be both a promising diagnostic marker and therapeutic target in the near future.

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