Salivary small extracellular vesicles reveal protein signatures in young patients with coronary artery disease.

Saliva-derived small extracellular vesicles (sEVs) are emerging as potential biomarkers for coronary artery disease (CAD). Early identification of these biomarkers is essential for effective management and improved patient outcomes. Our study aimed to isolate and characterize sEVs from saliva to identify non-invasive protein signatures in younger CAD patients. Saliva sEVs were obtained from 20 CAD patients aged 18-65 years, and 20 healthy controls matched for age and gender. The saliva exosome or sEVs isolation was performed using differential ultracentrifugation and sucrose density gradient methods, and we characterized the sEVs using transmission electron microscopy, scanning electron microscopy, and nanoparticle tracking analysis. Western blotting was done with exosome markers including Anti-Flotilin-1, Anti-TSG-101, and Anti-CD63. Differentially expressed proteins (DEPs) were identified through label-free LC-MS/MS Orbitrap and data was analyzed using Proteome Discoverer 3.0 and statistical analysis using MetaboAnalyst 6.0. Protein-protein interaction network, gene ontology, and pathways enrichment analysis were performed. We identified 506 proteins using label-free LC-MS/MS proteomic approaches, with 18 significant DEPs. Notable upregulated proteins included mainly Cystatin-S (CST1/CST2/CST4), Protein S100, alpha-amylase, and Gelsolin (GSN), while downregulated proteins included Serum albumin (ALB) and Apolipoprotein A1 (APOA1). These proteins are linked to inflammation and salivary secretions largely. For the first time, we present unique saliva sEVs protein signatures associated with CAD. Validation in larger cohorts may establish Cystatin S as a potential diagnostic biomarker for CAD.