A Bioorthogonal Ratiometric Fluorescence Nanoprobe for Imaging the Regulation of O‐GlcNAcylation in Atherosclerosis

Protein post-translational modification (PTM) plays a significant role in the progression of atherosclerosis (AS). However, the role of O-GlcNAcylation, as an important O-glycosylation modification, in the formation of foam cells remains unclear, limiting early diagnosis and mechanistic studies. Here, utilizing two bioorthogonal reactions, we developed a ratiometric fluorescent nanoprobe-based tracker for O-GlcNAcylation. This tracker (BORFI) enables dynamic tracing, relative quantification, and multi-scale in situ visualization of protein O-GlcNAcylation during cellular events, and images the regulation of O-GlcNAcylation during foam cell formation. Using BORFI, we successfully achieved spatiotemporal co-imaging of O-GlcNAcylation and reactive oxygen species (ROS), and found that there is a significant positive correlation between O-GlcNAcylation and ROS during the transition from macrophages to foam cells. Treatment with the OGT enzyme inhibitor (OSMI-1) to affect the O-GlcNAcylation level in foam cells effectively alleviates foam cell formation, accompanied by suppression of the overall increase in ROS levels, modulation of key cholesterol metabolism‑related proteins (downregulation of ACAT1 and upregulation of ABCA1), and downregulation of the scavenger receptor MSR1. Furthermore, BORFI was successfully applied to in situ imaging of the aorta in a mouse AS model, and we found that hyperglycemia participates in AS progression by specifically upregulating vascular O-GlcNAcylation.