Structural Behavior of the Endothelial Glycocalyx Is Associated With Pathophysiologic Status in Septic Mice: An Integrated Approach to Analyzing the Behavior and Function of the Glycocalyx Using Both Electron and Fluorescence Intravital Microscopy.

BACKGROUND: The endothelial surface layer (ESL) regulates vascular permeability to maintain fluid homeostasis. The glycocalyx (GCX), which has a complex and fragile ultrastructure, is an important component of the ESL. Abnormalities of the GCX have been hypothesized to trigger pathological hyperpermeability. Here, we report an integrated in vivo analysis of the morphological and functional properties of the GCX in a vital organ. METHODS: We examined the behavior of the ESL and GCX, using both electron microscopy (EM) and intravital microscopy (IVM). We also compared morphological changes in the ESL of mouse skin in a glycosidase-treated and control group. Combined approaches were also used to examine both morphology and function in a lipopolysaccharide-induced septic model and the pathophysiological features of leukocyte-endothelial interactions and in vivo vascular permeability. RESULTS: Using IVM, we identified an illuminated part of the ESL as the GCX and confirmed our observation using morphological and biochemical means. In septic mice, we found that the GCX was thinner than in nonseptic controls in both an EM image analysis (0.98 ± 2.08 nm vs 70.68 ± 36.36 nm, P< .001) and an IVM image analysis (0.36 ± 0.15 µm vs 1.07 ± 0.39 µm, P< .001). Under septic conditions, syndecan-1, a representative core protein of the GCX, was released into the blood serum at a higher rate in septic animals (7.33 ± 3.46 ng/mL) when compared with controls (below the limit of detection, P< .001). Significant increases in leukocyte-endothelial interactions, defined as the numbers of rolling or firm-sticking leukocytes, and molecular hyperpermeability to the interstitium were also observed after GCX shedding in vivo. CONCLUSIONS: Using IVM, we visualized an illuminated part of the ESL layer that was subsequently confirmed as the GCX using EM. Severe sepsis induced morphological degradation of the GCX, accompanied by shedding of the syndecan-1 core protein and an increase in leukocyte-endothelial interactions affecting the vascular permeability. Our in vivo model describes a new approach to deciphering the relationship between structural and functional behaviors of the GCX.

Glycocalyx and its involvement in clinical pathophysiologies.

Vascular hyperpermeability is a frequent intractable feature involved in a wide range of diseases in the intensive care unit. The glycocalyx (GCX) seemingly plays a key role to control vascular permeability. The GCX has attracted the attention of clinicians working on vascular permeability involving angiopathies, and several clinical approaches to examine the involvement of the GCX have been attempted. The GCX is a major constituent of the endothelial surface layer (ESL), which covers most of the surface of the endothelial cells and reduces the access of cellular and macromolecular components of the blood to the surface of the endothelium. It has become evident that this structure is not just a barrier for vascular permeability but contributes to various functions including signal sensing and transmission to the endothelium. Because GCX is a highly fragile and unstable layer, the image had been only obtained by conventional transmission electron microscopy. Recently, advanced microscopy techniques have enabled direct visualization of the GCX in vivo, most of which use fluorescent-labeled lectins that bind to specific disaccharide moieties of glycosaminoglycan (GAG) chains. Fluorescent-labeled solutes also enabled to demonstrate vascular leakage under the in vivo microscope. Thus, functional analysis of GCX is advancing. A biomarker of GCX degradation has been clinically applied as a marker of vascular damage caused by surgery. Fragments of the GCX, such as syndecan-1 and/or hyaluronan (HA), have been examined, and their validity is now being examined. It is expected that GCX fragments can be a reliable diagnostic or prognostic indicator in various pathological conditions. Since GCX degradation is strongly correlated with disease progression, pharmacological intervention to prevent GCX degradation has been widely considered. HA and other GAGs are candidates to repair GCX; further studies are needed to establish pharmacological intervention. Recent advancement of GCX research has demonstrated that vascular permeability is not regulated by simple Starling's law. Biological regulation of vascular permeability by GCX opens the way to develop medical intervention to control vascular permeability in critical care patients.

Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy.

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively-charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)-labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA-binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC-labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy.