A comprehensive proteomic profiling of urinary exosomes and the identification of early non-invasive biomarker in patients with coronary artery disease

Abstract

Urinary small extracellular vesicles or exosomes (uEVs) source could be an emerging trove of biomarkers in coronary artery disease (CAD). It is a chronic inflammatory disease having a long asymptomatic phase of fatty-fibrous development in arteries leading to angina, myocardial infarction, and death. Our study was aimed at identifying differential protein expression profiling of uEVs in CAD. We collected urine samples of CAD patients (n = 41) age 18–65 years and gender matched healthy controls (n = 41). We isolated uEVs using differential ultracentrifugation. Further, uEV samples were characterized by western blotting exosome markers (Flotillin, TSG, CD63, and CD9), nano tracking analysis, and transmission and scanning electron microscopy. A total of 508 proteins were identified by iTRAQ-based mass spectrometry. We observed protein expression levels of AZGP1, SEMG1/2, ORM1, IGL, SERPINA5, HSPG2, prosaposin, gelsolin, and CD59 were upregulated, and UMOD, KNG1, AMBP, prothrombin, and TF were downregulated. Protein-protein interactions, gene ontology and pathway analysis were performed to functionally annotate identified uEVs proteins. A novel uEVs differential protein signature is shown. On validating UMOD protein by ELISA in two clinically different CAD, stable-CAD patients had lower levels than healthy controls whereas recent myocardial infarction patients had lowest. Our findings suggest UMOD importance as early diagnostic biomarker. SignificanceCoronary artery disease is a chronic inflammatory disease caused by gradual deposition of cholesterol and fat along with other proteins to develop plaque inside arteries. This further leads to blockage of artery, heart attack and death. There are no identifiable early biomarkers to diagnose this. For the first time, we have identified the differentially expressed proteins isolated from non-invasive uEV of CAD patients compared to healthy controls by using MS Orbitrap and iTRAQ labelling of peptides. We have identified decreased levels of UMOD protein in CAD. These findings have been confirmed by ELISA. Furthermore, the levels of UMOD were observed as more highly decreased in recent myocardial infarction CAD patients, indicating the importance of this protein as an early diagnostic biomarker. Conclusively, our study represents a non-invasive urinary EVs trove of differentially expressed proteins in CAD. This will form a groundwork for understanding the pathophysiology of CAD and will help in future translational research utilizing uEVs.