DOI: 10.5061/dryad.6m905qfw8
title: Endothelial glycocalyx layer properties and its ability to limit leukocyte adhesion

description: The endothelial glycocalyx layer (EGL), consisting of long proteoglycans
 protruding from the endothelium, acts as a regulator of inflammation by
 preventing leukocyte engagement with adhesion molecules on the endothelial
 surface. The amount of resistance to adhesive events the EGL provides is
 the result of two properties: EGL thickness and stiffness. To determine
 these, we used an atomic force microscope to indent the surfaces of
 cultured endothelial cells with a glass bead and evaluated two different
 approaches for interpreting the resulting force-indentation curves. In one
 we treat the EGL as a molecular brush and in the other, as a thin elastic
 layer on an elastic half-space. The latter approach proved more robust in
 our hands and yielded a thickness of 110 nm and a modulus of 0.025 kPa.
 Neither value showed significant dependence on indentation rate. 
 The brush model indicated a larger layer thickness (~350 nm) but tended to
 result in larger uncertainties in the fitted parameters. The modulus of
 the endothelial cell was determined to be 3.0 – 6.5 kPa (1.5 – 2.5 kPa for
 the brush model) with a significant increase in modulus with increasing
 indentation rates.  For forces and leukocyte properties in the
 physiological range, a model of a leukocyte interacting with the
 endothelium predicts that the number of molecules within bonding range
 should decrease by an order of magnitude due to the presence of a 110
 nm-thick layer, and even further for a glycocalyx with larger thickness.
 Consistent with these predictions, neutrophil adhesion increased for
 endothelial cells with reduced EGL thickness because they were grown in
 the absence of fluid shear stress.  These studies establish a
 framework for understanding how glycocalyx layers with different thickness
 and stiffness limit adhesive events under homeostatic conditions and how
 glycocalyx damage or removal will increase leukocyte adhesion potential
 during inflammation.
description: In a typical
 experiment, 10 force-vs.-distance curves were obtained from a region of
 the cell near but not over the nucleus.  These curves were super-imposed
 and averaged (see Supplemental Materials) to obtain a single averaged
 indentation vs. force curve.  One of the more problematic aspects of
 interpreting AFM measurements on soft materials is determining exactly
 where the initial point of contact is between the indenter and the
 material.  In our case, we rationalize that the EGL is much softer than
 the cell, and so contributes little to the indentation at high forces. 
 Therefore, we follow the procedures of Simon et al. (26) and extrapolated
 data obtained at high forces  (in our case, F &gt; 2 nN) using a
 simple Hertz model to identify the location of the cell surface.  This
 zero-point location was held constant for the least-squares regressions of
 the different theoretical relationships to the averaged curves.  For the
 model regressions, there were three free parameters.  For the brush model,
 these were the brush coefficient
 (<i>kT</i>/<i>s</i><sup>3</sup>), the
 glycocalyx thickness and the modulus of the cell.  For the elastic-layer
 model, these were <i>t</i>, the thickness of the glycocalyx
 laye­r, <i>E</i><sub><i>GC</i></sub>,
 the modulus of the glycocalyx,
 <i>E</i><sub><i>cell</i></sub>, the
 modulus of the cell body.  We experimented with different values for the
 Poisson ratios for the EGL
 (<i>v</i><sub><i>G</i></sub>) and the
 cell (<i>v</i><sub><i>C</i></sub>). 
 (See Supplemental Material.)  The sensitivity of the fits to these
 parameters was not large, but a value of 0.3 for both
 <i>v</i><sub><i>G</i></sub> and
 <i>v</i><sub><i>C</i></sub> seemed to
 provide the greatest fidelity with the measured curves.  Therefore, the
 Poisson ratio for each of the layers was fixed at 0.3.  The values of the
 remaining parameters in the two-layer model were taken from the original
 modeling of Clifford and Seah (19): <i>P</i> = 2.25,
 <i>n</i> = 1.5, <i>m</i> = 2/3,
 <i>B<sub>L</sub></i> = 1.92 and
 <i>B<sub>s</sub></i> =
 0.22. 
description: DATA & FILE OVERVIEW Raw Indentation
 Data for the paper titled <i>Endothelial Glycocalyx Layer Properties
 and its ability to Limit Leukocyte Adhesion </i>is <i>
 </i>given and has been categorized according to the experimental
 parameters investigated in our paper.  The data has not
 been super-imposed and averaged (see Supplemental Materials) to obtain a
 single averaged indentation vs. force curve as stated in our
 methods.  Force Data is given in Newtons and is
 identified by the notation ImageXXXXForce_EXT and ImageXXXXForce_Ret for
 the extension and retract force curves respectively. 
 Piezo Sensor data is given in meters and is identified by the
 notation ImageXXXXZsnsr_EXT and ImageXXXXZsnsr_RET for the extension and
 retract indentation curves respectively. Deflection
 indentation data is in meters  and is identified by the notation 
 ImageXXXXDefl_Ext and ImageDefl_EXT for the extension and retract
 indentation curves respectively.