Distinct roles of glycocalyx components in regulating endothelial functions in a perfused three-dimensional human endothelium-on-a-chip

Increased degradation of the endothelial glycocalyx (EGX) is associated with cardiovascular disease. However, whether EGX impairment drives endothelial dysfunction or reflects disease severity remains unclear. Prior studies investigating EGX function primarily used two-dimensional (2D) endothelial cell cultures, which poorly mimic the endothelial microenvironment, particularly lacking luminal shear flow. To address these limitations, we leveraged a three-dimensional (3D) human endothelium-on-a-chip to examine the roles of EGX components, namely heparan sulfate (HS) and sialic acid (SA), in regulating vascular permeability and monocyte adhesion. EGX expression was markedly higher in perfused 3D human umbilical vein endothelial cells (HUVECs) cultures than in 2D cultures. In 3D HUVECs, tumor necrosis factor-alpha, a disruptor of endothelial function, did not reduce EGX expression, whereas dengue nonstructural protein 1 downregulated EGX. In 3D HUVECs, HS degradation by heparinase III significantly increased endothelial permeability to 70-kDa fluorescein isothiocyanate-dextran without inducing cytotoxicity, whereas SA cleavage by neuraminidase reduced vascular permeability. Interestingly, neither HS nor SA cleavage affected 3D human coronary artery endothelial cells (HCAECs) permeability. However, neuraminidase treatment significantly increased monocyte adhesion in both 3D HUVECs and HCAECs, an effect not observed in heparinase III-treated 3D endothelium from either vessel bed. These findings demonstrate that HS and SA play distinct roles in regulating endothelial barrier function and vascular inflammation in 3D human endothelium.