The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.
Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.
1. Methods
1.1 Cell Flow Chamber
A flow chamber, shown in Figure 1, was constructed so that cells could be grown under a shear of 1.0 Pa (10 dyn/cm2) and then transferred directly to an Asylum MFP3D AFM (Santa Barbara, CA).
where Q is the flow rate, τ is the shear stress, μ is the viscosity of the medium, assumed here to be 1.0 mPa (0.01 dyn*sec/cm2), h is the height and w is the width of the flow chamber.
1.2 Cell Culture
1.3 Cantilever Preparation and Cell Indentation
2. Indentation Theory
Indentation into an elastic half-space with a sphere of radius R can be described using Hertz theory where the force of indentation, F, is given by the equation:
Where δ is the indentation depth and E* is the reduced modulus of the material under test (Figure 3). In the case of an infinitely stiff indenter impinging a uniform elastic half-space, E* is given by the equation:
where E is the elastic modulus and ν is the Poisson ratio of the material. Recent work with polymer films has inspired the development of a two layer model for determining the modulus and thickness of thin films 1. We are applying this model to cell biology by treating the glycocalyx as a uniform thin soft film on the surface of the cell body. Using this model, the reduced modulus of the system becomes:
Where EGC is the modulus of the glycocalyx, Ecell is the modulus of the cell body, P, Q and n are constants which have been empirically determined from the polymer fits, and z is given by the equation:
Where t is the thickness of the glycocalyx layer. A schematic of these parameters is shown in Figure 3. The model has been shown to be an accurate way of determining the modulus and thickness of a thin film on stiffer substrate 1. This equation can be used to fit the curves obtained from indentation into cells to determine the modulus and thickness of the endothelial glycocalyx, as shown in Figure 4.
In a typical experiment, 20 force-vs-distance curves were obtained from a given region of the cell, typically in the perinuclear region, near, but not on, the nucleus (within ~2 μm). The curves were aligned to account for any sample drift over the duration of the measurement and then averaged to remove cantilever noise, as shown in Figure 4. The curves were analyzed and fit with the two layer model that was developed for determining the modulus and thickness of thin polymer films 1. From fits of the curves of 25 cells, we have determined that the modulus of the luminal layer is 0.7 ± 0.5 kPa and the thickness is 380 ± 50 nm, as shown in Figure 5. The modulus of the cell body is 16 ± 6 kPa. These values are in good agreement with previous measurements of the modulus of the cell body and thickness of the glycocalyx 5, 8.
Figure 1. Our cell culture device. Flow across the chamber was driven by gravity from reservoir A to C. Cells were plated in the closed chamber B which was held with magnets to the surface of a closed cell dish for an Asylum AFM (Asylum, Santa Barbara CA). Three-way valves on B enabled us to stop the flow. A silicone gasket created a flow channel 6.4 mm wide by 19 mm long by 0.4 mm high. The closed chamber B was easily removed and the closed cell dish moved directly into the AFM for experimentation. The height difference D was maintained by pumping fluid from reservoir C to A with a peristaltic pump (not shown). The whole assembly was placed in an incubator for the duration of cell culture.
Figure 2. Left: Image of a cantilever with a 2.4 μm bead (arrow) attached by a biotin-streptavidin linkage. Right: Monolayer of HUVECs grown under flow.
Figure 3. Geometry of the interaction of the bead of radius R indenting a distance δ into the cell. The glycocalyx, shown in green, has a modulus of EGC and a thickness t. The cell body has a modulus Ecell. The force applied by the bead to the cell, F, is measured and the force versus distance curve, shown in Figure 4, is obtained.
Figure 4. The averaged force versus distance curve for an indentation into a cell is plotted in red. An individual indentation is shown in the inset. Highlighted in the image are the contact point, where the cantilever first touches the luminal surface, the luminal layer, where the slope of the curve is dominated by the stiffness of the glycocalyx, and the cell body, where the slope is primarily a function of cell body modulus. The fitted curve from the two-layer indentation theory is shown in blue, and the fit for the simpler Hertz model for an elastic half space is shown by the dotted black line. There were four free parameters in the two-layer fits. Values determined for this particular fit were: cell modulus = 15.9 kPa, luminal layer modulus = 0.33 kPa, and luminal layer thickness = 420 nm. The fourth fitted parameter is the contact point with the glycocalyx. Positioning of the x-axis origin relative to the cell surface is arbitrary. Click here to view larger figure.
Figure 5. Histograms of the properties of 25 cells from the curve fits. Left: EGC was determined to be 0.7 ± 0.5 kPa. Right: The thickness of the glycocalyx was determined to be 380 ± 50 nm.
We used values calculated from the two-layer model and Hertz theory to model the interaction of a leukocyte circulating in the blood with the endothelial wall. We have calculated that a microvillus on the leukocyte with a diameter of 50 nm under a 10 pN load would indent approximately 150 nm into the glycocalyx, only a fraction of the total thickness. This indicates that the glycocalyx, with properties as measured in this experiment, is a significant barrier to cell-cell interaction and can be a large steric hindrance which the cells must overcome during the adhesion cascade during leukocyte adhesion.
In the model used here, we approximate the glycocalyx as an isotropic elastic structure. While we are unaware of any mechanical measurements to indicate this is not the case, what is known about the molecular structure of the glycocalyx suggests that this is likely an over-simplification. In fact, the glycocalyx is a complex and varied structure on the surface of cells. It consists of oriented molecular structures, lacks a well-defined outer boundary, and likely becomes denser close to the cell surface. Thus, while the two-layered elastic model used here provides insight into the relative stiffness of the glycocalyx observed by circulating cells, future studies could explore alternative mechanical descriptions that may account for its possible anisotropy and varying density in the thickness direction. It is also possible that the glycocalyx is not uniform across different regions of the cell surface. This would not have been evident from the present data set because all data presented here were taken in the central region of the cell around the nucleus.
The glycocalyx may also exhibit viscoelastic properties that were not investigated in this study. It has been observed that, under static conditions, red blood cells in capillaries can completely compress the glycocalyx, wheres circulating red blood cells do not 10. Forces generated by static red cells are likely to be extremely small (~3-10 Pa). This indicates the glycocalyx may be very soft in response to slow compression, but significantly stiffer during faster compression. Our measurements were performed at indentation rates of 1 μm/sec to simulate the stiffness a circulating cell may encounter, but further work to investigate the time-dependent properties is in progress.
AFM indentation has been used to directly measure the modulus and thickness of the endothelial glycocalyx in living cells. The measurements indicate that the glycocalyx can be a significant barrier to cell-cell contact and adhesion and likely serves as a key factor in regulating the adhesion cascade during inflammation.
The authors have nothing to disclose.
The authors would like to thank Elena Lomakina, Richard Bauserman, Margaret Youngman, Shay Vaknin, Jessica Snyder, Chris Striemer, Nakul Nataraj, Hung Li Chung, Tejas Khire, and Eric Lam for their assistance with this project. This project was funded by NIH #PO1 HL 018208.
Name of Reagent/Material | Company | Catalog Number | Comments |
McCoy’s Medium | Gibco | 16600-082 | |
Fetal Calf Serum | Hyclone | SH30070 | |
Endothelial Cell Growth Medium | Vec Technologies | MCDB-131 | |
Pooled Human Umbilical Vein Endothelial Cells | Vec Technologies | PHUVEC/T-25 | |
Sulfuric Acid | JT Baker | 9681-02 | |
Hydrogen Peroxide | VWR | BDH3742-1 | |
(3-aminopropyl)triethoxysilane | Aldrich | 440140-100ML | |
Isopropyl Alcohol | VWR | BDH8999-4 | |
Trypsin | Cellgro | 25-054-C1 | |
Hank’s Buffered Salt Solution | Gibco | 14175-095 | |
sulfo-NHS-LC-Biotin | Thermo Scientific | 21335 | |
Streptavadin beads | Dynabeads | 112.06D | |
MFP-3D AFM | Asylum Research | ||
Tipless Cantilevers | Nanoworld | ARROW-TL1-50 | |
Silhouette SD | Quickutz | Silhouette-SD | |
Silicone Rubber | Stockwell Elastomerics | SE50-RS | |
30 ml Syringes | Benton Dickinson | 309650 | |
18 gauge needles | Benton Dickinson | 305196 | |
Extension Sets | Hospira | 4429-48 | |
4 way valves | Teleflex | W21372 | |
Male/Female Port Caps | Smith’s Medical | MX491B | |
Peristaltic Pump | Watson-Marlow | 401U/D | |
Peristaltic Tubing | Watson-Marlow | 903.0016.016 | |
sterile filters | Pall Life Science | 4652 |