ESTIMATION OF ENDOTHELIAL GLYCOCALYX LAYER DEFORMATION IN RESPONSE TO FLUID SHEAR STRESS
Takeo Matsumoto1,2, Yutaka Takahashi1, Yasushi Owaki1, Kazuaki Nagayama1,3
1Nagoya Institute of Technology, Japan; 2Nagoya University, Japan; 3Ibaraki University, Japan
It has been pointed out that glycocalyx layer (GL) covering the surface of vascular endothelial cells (ECs) plays pivotal roles in their biomechanical responses. Since GL is made of sugar chains coming up from the endothelial surface, it may be sheared by fluid flow and this may amplify the torque applied to mechanoreceptors on the cell membrane. However, to our knowledge, there is no quantitative data on the deformation of GL in response to fluid shear stress. In this study, we estimated shear deformation of GL with three different methods.
The GL of a murine vascular endothelial cell line F-2 was stained with DyLight 488 Tomato Lectin. We plated the cells in a laboratory-made laminar flow chamber to apply fluid shear stress under a confocal laser scanning microscope. If the GL is sheared, its thickness may decrease. By measuring its thickness with a 100x oil-immersion objective, we found that it decreased from 840 nm to 750 nm in response to shear stress of 6 Pa, which corresponds to 4.5°/Pa. When the GL with photobleached area is sheared, its borderline may become blurred. By measuring fluorescent intensity distribution across the borderline before and after application of fluid shear stress (6 Pa) and analyzing it with a simple physical model, we obtained shear deformation of 5.1°/Pa. We then measured the displacements of quantum dots (Qdots) bound to the top of heparan sulfate and PECAM-1 on the cell membrane in response to fluid shear stress (2 Pa), and found that displacement was larger by 200 nm for Qdots bound to the heparan sulfate than that bound to PECAM-1. This value corresponds to 7.1°/Pa if the GL thickness is assumed to be 800 nm. All of these methods gave similar results. The shear deformation of the GL might be 5-10°/Pa.