Engineering Mechanical Microenvironments: Integration of Substrate and Flow Mechanics Reveals the Impact on the Endothelial Glycocalyx

The glycocalyx (GCX), a multicomponent coating on endothelial cells (ECs), plays a critical role in various cellular behaviors, including barrier formation, vasodilation, and mechanotransduction. Mechanical perturbations in the vascular environment, such as blood vessel stiffness, are sensed and transduced by ECs via the GCX. Hypertension-induced stiffness disrupts GCX-mediated mechanotransduction, leading to EC dysfunction and atherosclerotic cardiovascular diseases. Understanding GCX-regulated mechanotransduction necessitates an in vitro model that closely mimics in vivo conditions. Existing models are insufficient, prompting the development of the system described in this manuscript. Here, we report on a new system to model varying EC substrate stiffness under sustained physiological fluid shear stress, providing a realistic environment for comprehensive examination of EC function. Gelatin methacrylate (GelMA) substrates with stiffnesses of 5 kPa (physiological) and 10 kPa (pathological) were seeded with human umbilical vein ECs (HUVECs) and subjected to constant physiological shear stress (12 dyn/cm²) for 6 h. Analysis focused on heparan sulfate (HS), sialic acid (SA), hyaluronic acid (HA), syndecan-1 (SDC1), cluster of differentiation 44 (CD44), and Yes-associated protein (YAP). Compared to the 5 kPa conditions, HS coverage and thickness decreased at 10 kPa, indicating impaired barrier function and increased susceptibility to inflammatory agents. SA density increased despite decreased coverage, suggesting enhanced binding site availability for inflammatory recruitment. HA expression remained unchanged, but the amount of the HA core receptor, CD44, was found to be increased at 10 kPa. Consistent with previously published interactions between CD44 and YAP, we observed increased YAP activation at 10 kPa, as evidenced by increased nuclear translocation and decreased phosphorylation. These findings, bridging biomaterials and mechanobiology approaches, deepen our understanding of how mechanical stimuli influence the EC GCX function. The results underscore the potential of mechanotherapeutic strategies aimed at preserving vascular health by modulating the endothelial function.