Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy

Abstract

O'Callaghan R, Job KM, Dull RO, Hlady V. Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy. Am J Physiol Lung Cell Mol Physiol 301: L353-L360, 2011. First published June 24, 2011 doi:10.1152/ajplung.00342.2010.-The mechanical properties of endothelial glycocalyx were studied using atomic force microscopy with a silica bead (diameter ϳ18 m) serving as an indenter. Even at indentations of several hundred nanometers, the bead exerted very low compressive pressures on the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx and allowed for an averaging of stiffness in the bead-cell contact area. The elastic modulus of BLMVEC glycocalyx was determined as a pointwise function of the indentation depth before and after enzymatic degradation of specific glycocalyx components. The modulus-indentation depth profiles showed the cells becoming progressively stiffer with increased indentation. Three different enzymes were used: heparinases III and I and hyaluronidase. The main effects of heparinase III and hyaluronidase enzymes were that the elastic modulus in the cell junction regions increased more rapidly with the indentation than in BLMVEC controls, and that the effective thickness of glycocalyx was reduced. Cytochalasin D abolished the modulus increase with the indentation. The confocal profiling of heparan sulfate and hyaluronan with atomic force microscopy indentation data demonstrated marked heterogeneity of the glycocalyx composition between cell junctions and nuclear regions. atomic force microscopy; bovine lung microvascular endothelial cell; heparan sulfate; hyaluronan THE ENDOTHELIAL GLYCOCALYX is a polysaccharide-protein coating on the luminal surface of the vascular endothelium and forms a negatively charged, complex meshwork. The primary glycosaminoglycan (GAG) constituents of glycocalyx are heparan sulfates (HS), chondroitin sulfates, and hyaluronan (HA). The syndecan family of transmembrane proteoglycans and membrane-bound glypicans both carry HS and chondroitin sulfate side chains (29, 33), while HA is a nonsulfated GAG that is secreted into the pericellular space and is associated with other components of glycocalyx. In vivo, the glycocalyx is known to associate with blood proteins, such as fibrinogen (21) and albumin (2), that contribute to the permeability barrier of the vessel wall. Several important functions are assigned to the in vivo glycocalyx (37): 1) a molecular sieve and hydrodynamic barrier for transvascular exchange of macromolecules; 2) an exclusion layer preventing interactions of blood proteins and cells with the endothelial membrane, per se; 3) a modulator of leukocyte binding and rolling; and 4) a transducer of mechanical impulses to the intercellular cytoskeleton and associated signaling pathways.Glycocalyx shedding and degradation, in inflammation models, lead to impaired endothelial mechanotransduction of fluid shear stress (13, 25, 34), adhesion of platelets (36), and leukocytes (7,17,22). It also leads to the leakage of...

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