Multimodal Profiling Reveals Distinct Endothelial Activation Pathways Regulated by Flow and Heparan Sulfate.

Purpose: Atherosclerotic cardiovascular disease originates from endothelial dysfunction, characterized by a shift toward a pro-inflammatory state and increased production of reactive oxygen species (ROS). This dysfunction occurs under adverse mechanical conditions, such as blood flow oscillation, multi-directionality, recirculation, shear stress gradients, and low or stagnation flows. This study investigates how degradation of heparan sulfate (HS), a major component of the endothelial glycocalyx, drives the transition of endothelial cells from a functional, anti-inflammatory, and antioxidant phenotype under streamlined flow conditions to a dysfunctional, pro-inflammatory, and pro-oxidant phenotype when flow is stagnant. Pro-inflammatory and pro-oxidant endothelial behavior precedes atherosclerosis development. Methods: Human aortic endothelial cells were exposed to uniform shear stress (14 dynes/cm Results: Endothelial cells conditioned by 14 dynes/cm Conclusions: The endothelial glycocalyx is a protective, dynamic, and complex structure, with HS as a key component. This study demonstrates that intact HS mitigates endothelial dysfunction by suppressing inflammation linked to flow-dependent atherosclerosis, but not ROS production. Future research will focus on translating these findings into HS-targeted therapies for atherosclerotic cardiovascular disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-026-00884-3.