Proteomics of serum extracellular vesicles identifies a novel COPD biomarker, fibulin-3 from elastic fibres

概要

〔目的(Purpose)〕
In spite of massive investment, there are no specific biomarkers for a complex and heterogeneous chronic obstructive pulmonary disease (COPD). Obstacles to discover biomarkers involve the complexity of samples such as serum as well as the immaturity of proteomic technology. To overcome these major problems, we focused on proteins of serum extracellular vesicles (EVs). EVs have emerged as novel and important mediators in physiological and pathological situation. They are small membrane vesicles derived from multivesicular bodies. Most of the cells in the body release EVs, which contain a subset of proteins, lipids, and nucleic acids from the parental cells. Of importance, they have key roles in intercellular communication, both locally and systemically.
In order to obtain novel biomarkers for COPD, we performed quantitative high throughput non-targeted proteomics; and subsequent targeted proteomics, selected reaction monitoring.

〔方法ならびに成績(Methods/Results)〕
EVs from 10 patients with COPD and 6 healthy controls were analysed by tandem mass tag-based non-targeted proteomics, and those from elastase-treated mouse models of emphysema were also analysed by non-targeted proteomics. For validation, EVs from 23 patients with COPD and 20 healthy controls were validated by targeted proteomics.
By non-targeted proteomics, we identified 406 proteins, 34 of which were significantly up-regulated in patients with COPD. Of note, the EV protein signature from patients with COPD reflected inflammation and remodeling. We also identified 63 up- regulated candidates from 1956 proteins by analysing EVs isolated from mouse models. Combining human and mouse biomarker candidates, we validated 45 proteins by targeted proteomics, selected reaction monitoring. Notably, levels of fibulin-3, tripeptidyl-peptidase 2, fibulin-1. and soluble scavenger receptor cysteine-rich domain-containing protein were significantly higher in patients with COPD. Moreover, six proteins; fibulin-3, tripeptidyl-peptidase 2, UΤΡ--glucose-1- phosphate uridylyltransferase, CD81, CD177, and oncoprotein-induced transcript 3, were correlated with emphysema. Up­ regulation of fibulin-3 was confirmed by immunoblotting of EVs and immunohistochemistry in lungs. Strikingly, fibulin-3 knockout mice spontaneously developed emphysema with age, as evidenced by alveolar enlargement and elastin destruction.

〔総括(Conclusion)〕
We discovered potential pathogenic biomarkers for COPD using next-generation proteomics of EVs. This is a novel strategy for biomarker discovery and precision medicine.