Advanced glycation end product (AGE)-modified albumin does not enhance neutrophil extracellular trap formation and minimally impacts endothelial inflammation in large vessels.

AIMS: Advanced glycation end products (AGEs), particularly AGE-modified albumin (AGE-BSA), have been implicated in vascular inflammation and endothelial dysfunction, while neutrophil extracellular traps (NETs) are known to aggravate endothelial injury. Here, we investigated whether AGE-BSA directly promotes endothelial inflammatory and thrombotic activation, modulates neutrophil oxidative stress and NET formation, and may enhance NET-induced endothelial inflammation.

MATERIALS, METHODS, AND KEY FINDINGS: In human aortic and human umbilical vein endothelial cells, two different endotoxin-free AGE-BSA formulations did not or only minimally alter the expression or secretion of inflammatory (IL-6, IL-1β, CCL2) or thrombotic (VCAM-1, tissue factor) markers. In parallel, neutrophils from healthy donors were exposed to BSA or AGE-BSA, and neither basal nor PMA-stimulated NET formation nor reactive oxygen species (ROS) production were affected. Furthermore, endothelial cells stimulated with PMA-induced NETs generated in the presence of BSA or AGE-BSA showed comparable induction of inflammatory and pro-thrombotic genes, indicating that AGE-BSA does not modify NET-mediated endothelial activation. Flow cytometry revealed higher intracellular than surface RAGE levels in endothelial cells and a low surface expression in neutrophils, potentially limiting AGE-RAGE signaling at the plasma membrane.

SIGNIFICANCE: Collectively, these data demonstrate that, under the conditions tested, endotoxin-free AGE-BSA has no or minimal pro-inflammatory effect on human aortic or umbilical vein endothelial cells, neutrophil NET and ROS formation, nor NET-driven endothelial inflammation, potentially due to a limited surface RAGE availability or downstream signaling capacity. This challenges the view of an inherent pro-inflammatory effect of AGE-modified BSA and highlights the significance of important cell type- and context-specific contributors.