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

TL;DR

This study shows that heparan sulfate in the endothelial glycocalyx helps prevent inflammation in blood vessels under normal flow conditions, but not reactive oxygen species production.

Contribution

The study reveals that heparan sulfate specifically suppresses flow-dependent endothelial inflammation but not oxidative stress.

Findings

Intact heparan sulfate suppresses pro-inflammatory gene expression under normal flow conditions.

HS degradation leads to an inflammatory endothelial phenotype similar to stagnant flow conditions.

ROS production remains unaffected by HS degradation under normal flow.

Abstract

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.

Human aortic endothelial cells were exposed to uniform shear stress (14 dynes/cm2) to model healthy endothelium. Unhealthy conditions were simulated via static conditions (0 dynes/cm2) or enzymatic HS degradation using heparinase III. Endothelial cell phenotype was assessed using fluorescent labeling, confocal microscopy, Western blotting, and RNA sequencing.

Endothelial cells conditioned by 14 dynes/cm2 shear stress without heparinase III exhibited low expression of pro-inflammatory genes (HIF1A, VCAM1, and IL1B), minimal ROS production, and up-regulation of Kruppel-like transcription factors. Under the same flow conditions, HS degradation via heparinase III induced an inflammatory phenotype, resembling responses observed at 0 dynes/cm2 shear stress, while ROS levels remained largely unaffected.

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.

The online version contains supplementary material available at 10.1007/s12195-026-00884-3.

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