Abstract 1653: Sirolimus Accelerates Senescence of Endothelial Progenitor Cells through Telomerase Inactivation
Background: Sirolimus-eluting stent (SES) are commonly used to prevent in-stent restenosis but are not infrequently complicated by late angiographic stent thrombosis (LAST). On the other hand, endothelial progenitor cells (EPCs) play a significant role in the maintenance of endothelial integrity.
Aim: We examined whether sirolimus affects differentiation, proliferative activity, senescence, colony formation, and network formation in EPCs originated from mononuclear cells (MNCs).
Methods and Results: MNCs were isolated from peripheral blood of healthy volunteers. EPCs outgrew from the culture of MNCs. When MNCs were incubated with sirolimus at 0.01, 0.1, and 1 ng/mL for 4 day, sirolimus dose-dependently reduced the number of differentiated, adherent EPCs, as assessed by an in vitro culture assay. After ex-vivo cultivation, EPCs became senescent as determined by acidic β-galactosidase staining. When MNCs were treated with sirolimus, sirolimus dose-dependently accelerated the onset of EPCs senescene. RT-PCR analysis demonstrated that FK506-binding protein 12 (FKBP12), a receptor of sirolimus, was expressed in MNCs. To obtain an insight into the underlying downstream effects of sirolimus, we measured telomerase activity and the expression of p27kip1. Sirolimus decreased telomerase activity dose-dependently, which accompanied with down-regulation of the catalytic subunit, telomerase reverse transcriptase (TERT). Furthermore, sirolimus up-regulated the cell cycle inhibitor p27kip1. Having demonstrated that sirolimus accelerated the onset of senescence, we examined whether that translates into a decrease in proliferative activity and clonal expansion. Both MTS assay and BrdU incorporation assay have shown that sirolimus treatment significantly diminished the proliferative activity in EPCs. In addition, colony forming unit assay revealed that sirolimus dramatically decreased colony formation than that in control (no treatment). Finally, in a Matrigel assay, EPCs treated with sirolimus were shown to be less integrated into the network formation than control (no treatment).
Conclusion: The inhibitory effects of sirolimus on circulating EPCs potently may affect reendotheliazation after SES implantation.