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

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-alpha (100 ng/mL) for 1-24 hours in the presence or absence of everolimus using 0.5 pM concentration locally maintained by DES. EC activation was evaluated via the levels of IL-1 beta and IL-6 mRNAs with miR-155 expression by RT-qPCR as well as the nuclear translocation of nuclear factor kappa beta (NF-kappa B) detected by fluorescence microscopy. To investigate the transcriptional regulation of E-selectin and VCAM-1, TNF-alpha-induced enhancer RNA (eRNA) expression at p65-bound enhancers in the neighboring genomic regions of SELE and VCAM1 genes, including SELE-11 kappa b and VCAM1-10 kappa b, 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-alpha 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-alpha, while everolimus acted as a negative regulator of EC activation via inhibited translocation of NF-kappa B p65 subunit into cell nuclei, lowered eRNA expression at SELE and VCAM1 genes-associated enhancers and modulated expression of their posttranscriptional 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-kappa B p65 translocation causing decreased E-selectin and VCAM-1 expression at transcriptional and post-transcriptional level after DES.