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  • Sirolimus rapamycin and everolimus both of

    2022-06-15

    Sirolimus (rapamycin) and everolimus, both of which are mTOR inhibitors, are released by commonly used first- and second-generation DES. Considering the relatively high rates of the target vessel failure with currently available DES [12,14,16,45,46], there is a need for a novel drug strategy that prevents cell growth in and on the vessel wall to a much higher extent. In the present study, we compared the FasL-NO donor combination with two mTOR inhibitor drugs sirolimus and everolimus, in terms of their ability to prevent SMC growth and their effect on ECs. Sirolimus, at 10 nM concentration, has been previously shown to decrease the number of proliferating vascular SMC by 65% in 5 h and total cell number by 55% in 24 h, while decreasing proliferating ECs by 85% and total EC number by 55% [47]. Everolimus at 10 nM decreased SMC proliferation by 40% at 24 h after application [48]. Therefore, we chose 10 nM as the working concentration for sirolimus and everolimus to compare their effects on SMCs with the combination of FasL and NO donor DetaNONOate, 400 ng/mL and 0.1 mM, respectively. We showed that, at 48 h after addition to the cell culture medium, the FasL-NO donor combination decreases total SMC number by 93 ± 2.1% by triggering apoptosis, while sirolimus and everolimus decreased total cell number by 36 ± 9.8% and 53 ± 13.8%, respectively. At the end of 48 h, the percentages of proliferating E7080 were 5.5%, 10.6% (not significantly different than the control group) and 1.4% for sirolimus, everolimus, and the FasL-NO donor combination. Notably, the FasL-NO donor combination used here had minimal effect on EC viability, proliferation and function, unlike sirolimus and everolimus. mTOR inhibitors are known to reduce EC proliferation and migration [49] and eNOS expression [12,39] in ECs. Non-complete or non-functional de novo endothelium creates the need for long-term dual antiplatelet therapy, which in turn leads to increased bleeding/stroke risk. Despite the low rates of definite thrombosis reported in long-term studies, the significant target vessel revascularization [46] and treatment failure may be related to non-functional endothelium. Here we showed that FasL-NO donor combination (400 ng/mL and 0.1 mM, respectively) resulted in more than 90% reduction in SMC numbers, and had no significant effect on arterial EC viability or proliferation. This same dose of FasL-NO reduced the eNOS gene expression by 27% in cultured ECs, while both sirolimus and everolimus caused more than a 67% decrease. Therefore, FasL-NO donor combination is an outstanding candidate drug combination to inhibit intimal hyperplasia and restenosis, while minimizing the damage to endothelial restoration. While some prior studies have shown that NO donors promote EC survival by increasing resistance to external apoptotic stimuli or increasing vascular endothelial growth factor synthesis [50,51], others report a negative effect on EC proliferation or adhesion protein expression at higher concentrations of NO donors [[52], [53], [54]]. Our results showed that FasL alone can inhibit SMC growth significantly, and even low concentration of NO donor (0.05 mM DetaNONOate) can enhance this effect significantly, triggering Caspase 8 cleavage (Fig. S3 and Fig. 2). In contrast, even the combination of FasL and higher DetaNONOate concentration (0.2 mM) did not cause Fas-mediated apoptosis in ECs. However, 0.2 mM Detanonoate decreased EC proliferation significantly, independent of FasL (Fig. S4). The dip-coated stents were able to trigger significantly more SMC apoptosis in the stent proximity, while sparing a confluent EC layer in the lumen (Fig. 8). Yet, it is important to note that the doses that are delivered to the vessel wall must be determined precisely for subsequent translational in vivo studies. In the present study, we demonstrated the unique ability of the FasL-NO donor combination to differentially effect SMCs and ECs. However, further research is needed to model the effective release doses and the diffusion kinetics of NO donor- and FasL, with the goal of increasing SMC apoptosis significantly in the intima without affecting EC viability.