CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling

Long noncoding RNAs have emerged as biomarkers and regulators of cardiovascular disease. However, the expression pattern of circulating extracellular vesicle (EV)-incorporated lncRNAs in patients with coronary artery disease (CAD) is still poorly investigated. A human lncRNA array revealed that certain EV-lncRNAs are significantly dysregulated in CAD patients. Circulating small EVs (sEVs) from patients with (n=30) or without (n=30) CAD were used to quantify PUNISHER, GAS5, MALAT1, and H19 RNA levels. PUNISHER (p=0.002) and GAS5 (p=0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Fluorescent labeling and RT-qPCR of sEVs demonstrated that functional PUNISHER was transported into the recipient cells. Mechanistically, the RNA-binding protein, hnRNPK, interacts with PUNISHER, regulating its loading into sEVs. Knockdown of PUNISHER abrogated the EV-mediated effects on EC migration, proliferation, tube formation, and sprouting. Angiogenesis-related gene profiling showed that the expression of VEGFA RNA was significantly increased in EV-recipient cells. Protein stability and RNA-immunoprecipitation indicated that the PUNISHER-hnRNPK axis regulates the stability and binding of VEGFA mRNA to hnRNPK. Loss of PUNISHER in EVs abolished the EV-mediated promotion of VEGFA gene- and protein expression. Intercellular transfer of EV-incorporated PUNISHER promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.