## Endothelial Glycocalyx

### Abstract

The glycocalyx is a polysaccharide structure that protrudes from the body of a cell. It is primarily conformed of glycoproteins and proteoglycans, which provide communication, electrostatic charge, ionic buffering, permeability, and mechanosensation‐mechanotransduction capabilities to cells. In blood vessels, the endothelial glycocalyx that projects into the vascular lumen separates the vascular wall from the circulating blood. Such a physical location allows a number of its components, including sialic acid, glypican‐1, heparan sulfate, and hyaluronan, to participate in the mechanosensation‐mechanotransduction of blood flow‐dependent shear stress, which results in the synthesis of nitric oxide and flow‐mediated vasodilation. The endothelial glycocalyx also participates in the regulation of vascular permeability and the modulation of inflammatory responses, including the processes of leukocyte rolling and extravasation. Its structural architecture and negative charge work to prevent macromolecules greater than approximately 70 kDa and cationic molecules from binding and flowing out of the vasculature. This also prevents the extravasation of pathogens such as bacteria and virus, as well as that of tumor cells. Due to its constant exposure to shear and circulating enzymes such as neuraminidase, heparanase, hyaluronidase, and matrix metalloproteinases, the endothelial glycocalyx is in a continuous process of degradation and renovation. A balance favoring degradation is associated with a variety of pathologies including atherosclerosis, hypertension, vascular aging, metastatic cancer, and diabetic vasculopathies. Consequently, ongoing research efforts are focused on deciphering the mechanisms that promote glycocalyx degradation or limit its syntheses, as well as on therapeutic approaches to improve glycocalyx integrity with the goal of reducing vascular disease. © 2022 American Physiological Society. Compr Physiol 12: 1–31, 2022.

 Figure 1. Schematic illustration of the structural components of a small artery. The vascular wall consists of three distinct layers: the tunica externa (adventitia), the tunica media and the tunica intima. The adventitia is composed of collagen‐rich connective tissue containing fibroblasts and perivascular nerves. The tunica media contains mainly vascular smooth muscle cells and extracellular matrix (ECM) components including elastic laminas between smooth muscle layers as well as an internal elastic lamina that separates the tunica media and from the intima. The tunica intima contains a basement membrane and a monolayer of endothelial cells (endothelium) that synthesizes and anchors all components of the endothelial glycocalyx. Modified, with permission, from Martinez‐Lemus LA, 2012 182. Figure 2. Schematic representation of the endothelial glycocalyx components. The endothelial glycocalyx is prominently present on the apical surface of the vascular endothelium. It has a brush‐like structure conformed of glycoproteins (e.g., CD44), some of which have attached glycosaminoglycan chains (e.g., heparan sulfate) that together form proteoglycan components (e.g., syndecan‐1). The glycocalyx components can be directly anchored to the cell membrane via transmembrane domains or via covalent links to molecules that associate with the endothelial plasmalemma. These anchoring associations include those with caveolin (Cav), protein kinase C (PKC), and other plasma membrane and intracellular molecules. They allow the glycocalyx to participate in the mechanotransduction of physical forces and the subsequent activation of intracellular pathways. Such pathways include the formation of calcium‐calmodulin (Ca‐Cam) complexes, the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS), the modulation of cytoskeletal structures, and the formation of endothelial focal adhesions. Overall the characteristics of the vascular endothelium are modulated by shedding and adsorption processes that change the abundance of each glycocalyx component present on the cell surface. Figure 3. Schematic representation of the endothelial glycocalyx exposed to blood flow‐generated shear stress and the signaling cascade that results in shear stress‐generated nitric oxide (NO). The endothelial glycocalyx is exposed to laminar, oscillatory or disturbed blood flow patterns that deform, stimulate or shed glycocalyx components resulting in diverse outcomes, including the activation of endothelial NO synthase (eNOS), the production of NO and the subsequent induction of vasodilation. Figure 4. Illustration of the forces that drive vascular permeability and their relationship with the endothelial glycocalyx. Under nonpathological conditions, the intact endothelial glycocalyx contributes significantly to the vascular permeability process as a physical and electrostatic barrier. Although the filtering of molecules is predominantly a function of cell‐cell adherens junctions and paracellular tight junctions, the negatively charged bush‐like structure of the glycocalyx, prevents the extravasation of macromolecules greater than approximately 70 kDa, as well as that of cationic molecules. Figure 5. Two of the major sialic acid residues present in the endothelial glycocalyx are N‐acetylneuraminic acid and N‐glycolylneuraminic acid, which vary on their linkage to sugar moieties present in glycoprotein members of the glycocalyx. Figure 6. The endothelial glycocalyx is negatively charged. This allows for the formation of a diffuse electrostatic barrier that separates the endothelium from other negatively charged blood components, such as red blood cells. Figure 7. Schematic representation of the perfused boundary region (PBR) that allows for assessment of in vivo endothelial glycocalyx thickness. Flowing red blood cells (RBC) form a column as they travel within the vascular lumen. Blood velocity and the electrostatic charges of the endothelial glycocalyx as well as those of RBCs form a PBR, where only a few RBCs occasionally penetrate. The size of the median RBC column width subtracted from the vascular luminal diameter represents a measure of the PBR and the thickness of the endothelial glycocalyx. Modified, with permission, from Dane MJ, et al., 2015 56. Figure 8. Schematic representation of the mechanisms that promote endothelial glycocalyx degradation. The endothelial glycocalyx undergoes a constant process of synthesis or adsorption and degradation or shedding. Degradation occurs in response to glycosaminoglycans (GAG) oxidation by reactive oxygen species (ROS) such as nitric oxide (NO), superoxide ($O2•$), and H2O2, as well as by reactive nitrogen species (RNS) such as peroxynitrite (ONOO), N2$O•$ or N2O3. These reactive molecules are produced by multiple cellular components including mitochondria, endothelial NO synthase (eNOS), xanthine oxidase, and the NADPH oxidases (NOXes). Glycocalyx degradation also occurs by proteolysis or shedding mediated by enzymes such as the matrix metalloprotienases (MMPs) and the a disintegrin and metalloproteinases (ADAMs), amongst others. Glycocalyx degradation in turn increases vascular permeability and allows for exposure of proinflammatory receptors (e.g., P‐Selectin, ICAM, PE‐CAM, V‐CAM) that promote leukocyte rolling and adhesion to the vascular endothelium. Figure 9. Homeostasis of the endothelial glycocalyx relies on an appropriate balance between glycocalyx degradation or shedding and biosynthesis or adsorption. However, numerous conditions tilt the balance toward glycocalyx degradation. Such conditions include disturbed blood flow patterns, hypertension, aging and diabetes. Exacerbated glycocalyx degradation exposes the vasculature to infiltration and adhesion of pathogens and pro‐inflammatory molecules, thus leading to vascular inflammation, atherosclerosis, and endothelial dysfunction. (A) schematic depiction of a healthy endothelial glycocalyx (left panel) and one in which disturbed blood flow, hypertension, aging or diabetes has favored its degradation. (B) Confocal microscope generated images of an isolated small artery showing the endothelial glycocalyx [stained with wheat germ agglutinin (WGA), green] and vascular cell nuclei [stained with 4′,6‐diamidino‐2‐phenylindole (DAPI), blue] before (left) and after (right) enzymatic degradation of the glycocalyx. Figure 10. The endothelial glycocalyx is very fragile and difficult to measure. Today two commonly used techniques that provide ex vivo direct measurements of the in situ endothelial glycocalyx characteristics include atomic force microscopy (AFM) and transmitted electron microscopy (TEM). (A) AFM allows for the measurement of glycocalyx length and stiffness as assessed by the force curves generated when an AFM cantilever approaches and deforms the glycocalyx structures of en face isolated vascular preparations or cultured endothelial cells. (B) TEM allows for visualization of endothelial glycocalyx structures at the highest resolution currently available in microscopy, albeit, requiring complex sample preparation (Bar = 200 nm).