Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms after drug-eluting coronary stenting

• Published in: PloS one 2018-06, Vol.13 (6), p.e0197890-e0197890
• Main Authors: Fejes, Zsolt, Czimmerer, Zsolt, Szük, Tibor, Póliska, Szilárd, Horváth, Attila, Balogh, Enikő, Jeney, Viktória, Váradi, Judit, Fenyvesi, Ferenc, Balla, György, Édes, István, Balla, József, Kappelmayer, János, Nagy, Jr, Béla
• Format: Article
• Language: English
• Subjects: Activation; Attenuation; Biochemistry; Bioinformatics; Biology and Life Sciences; Biomarkers; Cardiology; Cell activation; Cell adhesion & migration; Cell cycle; Comparative analysis; Complications; Coronary artery; Coronary vessels; Coronary Vessels - cytology; Down-Regulation - drug effects; Drug delivery; Drug-Eluting Stents; E-selectin; E-Selectin - blood; E-Selectin - metabolism; Endothelial cells; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Endothelium; Enhancers; Everolimus - pharmacology; Fluorescence; Fluorescence microscopy; Gene expression; Gene regulation; Genes; Health aspects; Humans; Implants; Inhibitor drugs; Interleukin 6; Interleukin-1beta - metabolism; Interleukin-6 - metabolism; Internal medicine; Laboratories; Medicine; Medicine and Health Sciences; MicroRNAs; MicroRNAs - genetics; miRNA; Molecular biology; Neutrophils; NF-κB protein; Nuclear transport; Nuclei (cytology); Patients; Pharmaceutical sciences; Pharmacy; Post-transcription; Regulatory mechanisms (biology); Restenosis; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; Stents; Surgical implants; Thrombosis; Transcription (Genetics); Transcription, Genetic - drug effects; Translocation; Tumor necrosis factor; Tumor Necrosis Factor-alpha - pharmacology; Tumor necrosis factor-TNF; Tumor necrosis factor-α; Umbilical vein; Vascular cell adhesion molecule 1; Vascular Cell Adhesion Molecule-1 - blood; Vascular Cell Adhesion Molecule-1 - genetics; Veins & arteries
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0197890

We previously found higher level of endothelial cell (EC) activation in patients who suffered from in-stent restenosis after bare-metal stenting compared to subjects who underwent drug-eluting stenting (DES) showing no complications. Here we investigated the potential transcriptional and post-transcriptional regulatory mechanisms by which everolimus attenuated EC activation after DES. We studied the effect of everolimus on E-selectin (SELE) and VCAM1 mRNA levels when human coronary artery (HCAECs) and human umbilical vein ECs were challenged with recombinant TNF-α (100 ng/mL) for 1-24 hours in the presence or absence of everolimus using 0.5 μM concentration locally maintained by DES. EC activation was evaluated via the levels of IL-1β and IL-6 mRNAs with miR-155 expression by RT-qPCR as well as the nuclear translocation of nuclear factor kappa beta (NF-κB) detected by fluorescence microscopy. To investigate the transcriptional regulation of E-selectin and VCAM-1, TNF-α-induced enhancer RNA (eRNA) expression at p65-bound enhancers in the neighboring genomic regions of SELE and VCAM1 genes, including SELE_-11Kb and VCAM1_-10Kb, were measured in HCAECs. Mature and precursor levels of E-selectin and VCAM-1 repressor miR-181b were quantified to analyze the post-transcriptional regulation of these genes in HCAECs. Circulating miR-181b was analyzed in plasma samples of stented subjects by stem-loop RT-qPCR. TNF-α highly elevated E-selectin and VCAM-1 expression at transcriptional level in ECs. Levels of mature, pre- and pri-miR-181b were repressed in ECs by TNF-α, while everolimus acted as a negative regulator of EC activation via inhibited translocation of NF-κB p65 subunit into cell nuclei, lowered eRNA expression at SELE and VCAM1 genes-associated enhancers and modulated expression of their post-transcriptional repressor miR-181b. Significant negative correlation was observed between plasma miR-181b and soluble E-selectin and VCAM-1 in patients. In conclusion, everolimus attenuates EC activation via reduced NF-κB p65 translocation causing decreased E-selectin and VCAM-1 expression at transcriptional and post-transcriptional level after DES.