Coronary Artery Disease Increases the Long Noncoding RNA PUNISHER in Small Extracellular Vesicles and Regulates Endothelial Cell Function via Vesicular Shuttling

Mohammed Rabiul Hosen (Bonn)1, Q. Li (Bonn)1, Y. Liu (Bonn)1, A. Zietzer (Bonn)1, P. R. Goody (Bonn)1, E. Latz (Bonn)2, F. Jansen (Köln)3, N. Werner (Trier)4, G. Nickenig (Bonn)1

1Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; 2Institute of Innate Immunity University Hospital Bonn Bonn, Deutschland; 3Gemeinschaftspraxis Kardiologie Köln am Neumarkt Köln, Deutschland; 4Krankenhaus der Barmherzigen Brüder Trier Innere Medizin III Trier, Deutschland



Aims: Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular disease. Circulating lncRNAs can be incorporated into extracellular vesicles (EVs). Our aim was to study the expression pattern of circulating EV-incorporated lncRNAs in patients with and without coronary artery disease (CAD). The regulation of the lncRNA PUNISHER was further assessed as an intercellular messenger in endothelial cell (EC) biology.

Methods and results: Circulating small EVs (sEVs) were isolated from the plasma of patients using ultracentrifugation. Electron microscopy, western blot, and NTA were used to determine the size and origin of the isolated EVs. PCR-based human lncRNA array analysis revealed that certain EV-lncRNAs are significantly different in patients with CAD compared to patients without CAD. To validate the lncRNA array results, 60 patients with (n=30) or without (n=30) CAD were prospectively studied. Four atherosclerosis-related lncRNAs (PUNISHER, GAS5, MALAT1, and H19) were quantified in the circulating EVs by using real-time quantitative PCR (RT-qPCR). Among these, PUNISHER (p=0.002) and GAS5 (p=0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Analysis of RNA degradation revealed that circulating PUNISHER is mainly incorporated within small EVs. In vitro, atherosclerotic stimuli (oxLDL or TNF-a treatment) caused an upregulation of PUNISHER levels in human coronary artery endothelial cells (HCAECs) and in the corresponding sEVs. Labeling of sEVs followed by RT-qPCR demonstrated that functional PUNISHER was transported into the recipient ECs, which accelerated cell migration, proliferation, and tube formation. Mechanistically, the RNA-binding protein hnRNPK was identified by an RNA immunoprecipitation (RIP) assay as an interaction partner of PUNISHER, regulating its loading into small EVs. Knockdown of PUNISHER abrogated the EV-mediated effects on EC migration, proliferation, and tube formation. PCR-based gene profiling showed that the expression of VEGF RNA was significantly increased in ECs by treatment with sEVs. Protein stability and RNA-immunoprecipitation followed by RT-qPCR analysis indicated that the PUNISHER-hnRNPK axis regulates the stability and binding of VEGFA mRNA to hnRNPK. Knockdown of PUNISHER in EVs abrogated the EV-mediated promotion of VEGFA gene- and protein expression.

Conclusion: The circulating lncRNA PUNISHER is increased in CAD patients. Intercellular transfer of EV-incorporated PUNISHER promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.

Keywords: Extracellular Vesicles, Endothelial Cells, Long Noncoding RNA, Coronary Artery Disease

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