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Endothelial glycocalyx layer properties and its ability to limit leukocyte adhesion

Citation

Delgadillo, Luis (2020), Endothelial glycocalyx layer properties and its ability to limit leukocyte adhesion, Dryad, Dataset, https://doi.org/10.5061/dryad.6m905qfw8

Abstract

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.

Methods

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 > 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 (kT/s3), the glycocalyx thickness and the modulus of the cell.  For the elastic-layer model, these were t, the thickness of the glycocalyx laye­r, EGC, the modulus of the glycocalyx, Ecell, the modulus of the cell body.  We experimented with different values for the Poisson ratios for the EGL (vG) and the cell (vC).  (See Supplemental Material.)  The sensitivity of the fits to these parameters was not large, but a value of 0.3 for both vG and vC 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): P = 2.25, n = 1.5, m = 2/3, BL = 1.92 and Bs = 0.22. 

Usage Notes

DATA & FILE OVERVIEW

Raw Indentation Data for the paper titled Endothelial Glycocalyx Layer Properties and its ability to Limit Leukocyte Adhesion is  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.