# 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.

## Key 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.