Thickness and stiffness of the eGC were determined using the Atomic Force Microscope (AFM) nanoindentation technique. Preservation of the endothelial cell layer on aorta preparations was approved by immunostaining of PECAM-1/CD31 (Figure 1 ). Figure 2 A, B illustrates the basic principles of this method. By using a Multimode AFM (Veeco, Mannheim, Germany) with a feedback-controlled heating device (Veeco) measurements were performed at 37°C as described previously [29 (link)].
In brief, the central component of the AFM is a very sensitive mechanical nanosensor – a triangular cantilever with a mounted spherical tip (here: electrically uncharged polystyrene, diameter = 10 µm, Novascan, Ames, IA, USA) that is utilized to periodically indent the cells. A spherical tip was used for this AFM approach instead of a sharp tip because of a larger interaction area between tip and sample that decreases the effective pressure and results in less mechanical noise [30 (link)]. The cantilever functions as a soft spring (spring constant = 11 pN/nm). The xyz-position of the tip is precisely controlled by a piezo-element (Figure 2 A ). A laser beam is reflected by the gold-coated backside of the cantilever to a position-sensitive quadrupled photodiode allowing measurements of the cantilever deflection (V). Determination of the spring constant (Kcant) by the thermal tuning method and measurement of the deflection sensitivity (α) of the cantilever on bare glass coverslips facilitate the calculation of the force (F) acting on the cantilever and, in turn, the force exerted by the cantilever to the sample.
Since the piezo displacement (xpiezo) and the deflection sensitivity (α) are known, the indentation depth (deformation) of the sample (xsample) can be calculated.
For reasons of readability the indentation depth is hereafter called “thickness”. It should be noted that the indentation depth rather represents an apparent thickness, rather than the exact anatomical thickness.
Force indentation curves of a single cell were obtained by plotting the force (F) necessary to indent the cell (indentation depth, xsample). The sample stiffness can be derived from Hook´s law.
The stiffness (K) is the mechanical resistance of a sample against a defined deformation (e.g. indentation). K depends strongly on the indentation depth and the location, because cells contain a variety of substructures and organelles. The experimental parameters including an indentation velocity of 1 µm/s, a loading force of approximately 400 pN, an indentation frequency in the range of 0.25 - 0.5 Hz, a ramp size of 2 µm, a trig threshold of 35 nm and a tip velocity of 0.5 - 1 µm/s.
Previous experiments, using 1 µm AFM-tips, showed that the glycocalyx thickness is somewhat variable [29 (link),31 (link)]. Since we were interested in the overall condition of the glycocalyx and especially in its changes induced by different stimuli, we here chose larger tips (10 µm), as they indent a larger area. Thus they provided “more averaged” results and enabled us to avoid the data being influenced by the spatial distribution of the eGC thickness. All measurements were performed in HEPES-buffered solution [standard composition in millimolars: 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 5 Glucose, 10 HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.4] supplemented with 1% FCS in order to prevent eGC collapse [32 (link)].
Light microscopy was used to ensure that the tip position of the mechanical nanosensor was located neither at the nuclear, nor at the junctional region of cultured endothelial cells. However, this approach was not feasible in (thick) explanted aortas due to the lack of transparency of sub-endothelial layers such as the Tunica media and T. externa.
Figure 3 A, B show typical force indentation curves of an untreated as well as heparinase-treated aortic endothelial cell (“overview mode”). Each force indentation curve was then analyzed separately with a higher magnification (“working mode”) by using a linear approximation for determination of the eGC nanomechanics (Figure 3 C ).
In brief, the central component of the AFM is a very sensitive mechanical nanosensor – a triangular cantilever with a mounted spherical tip (here: electrically uncharged polystyrene, diameter = 10 µm, Novascan, Ames, IA, USA) that is utilized to periodically indent the cells. A spherical tip was used for this AFM approach instead of a sharp tip because of a larger interaction area between tip and sample that decreases the effective pressure and results in less mechanical noise [30 (link)]. The cantilever functions as a soft spring (spring constant = 11 pN/nm). The xyz-position of the tip is precisely controlled by a piezo-element (
Since the piezo displacement (xpiezo) and the deflection sensitivity (α) are known, the indentation depth (deformation) of the sample (xsample) can be calculated.
For reasons of readability the indentation depth is hereafter called “thickness”. It should be noted that the indentation depth rather represents an apparent thickness, rather than the exact anatomical thickness.
Force indentation curves of a single cell were obtained by plotting the force (F) necessary to indent the cell (indentation depth, xsample). The sample stiffness can be derived from Hook´s law.
The stiffness (K) is the mechanical resistance of a sample against a defined deformation (e.g. indentation). K depends strongly on the indentation depth and the location, because cells contain a variety of substructures and organelles. The experimental parameters including an indentation velocity of 1 µm/s, a loading force of approximately 400 pN, an indentation frequency in the range of 0.25 - 0.5 Hz, a ramp size of 2 µm, a trig threshold of 35 nm and a tip velocity of 0.5 - 1 µm/s.
Previous experiments, using 1 µm AFM-tips, showed that the glycocalyx thickness is somewhat variable [29 (link),31 (link)]. Since we were interested in the overall condition of the glycocalyx and especially in its changes induced by different stimuli, we here chose larger tips (10 µm), as they indent a larger area. Thus they provided “more averaged” results and enabled us to avoid the data being influenced by the spatial distribution of the eGC thickness. All measurements were performed in HEPES-buffered solution [standard composition in millimolars: 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 5 Glucose, 10 HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.4] supplemented with 1% FCS in order to prevent eGC collapse [32 (link)].
Light microscopy was used to ensure that the tip position of the mechanical nanosensor was located neither at the nuclear, nor at the junctional region of cultured endothelial cells. However, this approach was not feasible in (thick) explanted aortas due to the lack of transparency of sub-endothelial layers such as the Tunica media and T. externa.
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