Circulating long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators in cardiovascular diseases. Whether small extracellular vesicles (sEV)-incorporated lncRNAs expression is regulated in coronary artery disease (CAD) is largely unknown. We aimed to explore the expression of plasma sEV-lncRNAs in patients with and without CAD as well as elucidate specific cellular function of sEV-incorporated lncRNA PUNISHER (also known as AGAP2-AS1) in vascular endothelial cells.
Methods and results:
We quantified the expression level of 13 cardiac- or atherosclerosis-related lncRNAs by quantitative real-time PCR (qRT-PCR) in plasma sEV from 6 stable CAD, 6 ACS and 6 non-CAD (NCAD) patients. 4 lncRNAs showing stable expression (PUNISHER, GAS5, MALAT1, and H19) were further evaluated in the validation study including 65 stable CAD, 43 ACS and 42 NCAD patients. Among those, PUNISHER was significantly increased in patients with stable CAD and ACS compared to NCAD. Area under the curve (AUC) value was 0.7 for stable CAD and 0.8 for ACS respectively, suggesting plasma sEV-PUNISHER as a potential diagnostic biomarker for CAD. In vitro, atherosclerotic stimuli consisting of oxLDL or TNF-a treatment upregulated PUNISHER level in human coronary artery endothelial cell (HCAEC) and in corresponding sEV in a dose-dependent way. Mechanistically, RNA immunoprecipitation (RIP) assay identified hnRNPK as an interaction partner of PUNISHER regulating its packaging into sEV. In vitro, endothelial-derived sEV were internalized into recipient ECs and transported incorporated PUNISHER into recipient cells promoting an angiogenic response. Mechanical downstream analysis using PCR array showed that sEV-mediated PUNISHER transfer altered expression of vascular endothelial growth factor A (VEGFA) in recipient ECs.
This translational study highlights sEV-incorporated lncRNA PUNISHER as biomarker and effector in vascular disease.