Sirolimus accelerates senescence of endothelial progenitor cells through telomerase inactivation.
BACKGROUND
Sirolimus-eluting stent (SES) is commonly used to prevent in-stent restenosis but is not infrequently complicated by late angiographic stent thrombosis (LAST). On the other hand, circulating endothelial progenitor cells (EPCs) play a significant role in the maintenance of endothelial integrity.


OBJECTIVE
We examined whether sirolimus affects differentiation, proliferative activity, senescence, colony formation, and network formation in EPCs originated from mononuclear cells (MNCs).


RESULTS
MNCs were isolated from peripheral blood of healthy volunteers. EPCs outgrew from the culture of MNCs in the presence of vascular endothelial growth factor. When MNCs were incubated with sirolimus at 0.01, 0.1, and 1 ng/ml for 4 days, 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 beta-galactosidase staining. When MNCs were treated with sirolimus, sirolimus dose-dependently accelerated the onset of EPCs senescence. 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 p27(kip1). Sirolimus decreased telomerase activity dose-dependently, which was accompanied with down-regulation of the catalytic subunit, telomerase reverse transcriptase (TERT). Furthermore, sirolimus up-regulated the cell cycle inhibitor p27(kip1). Having demonstrated that sirolimus accelerated the onset of senescence, we examined whether that translated 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 as compared to 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).


CONCLUSIONS
The inhibitory effects of sirolimus on circulating EPCs potently may affect re-endotheliazation after SES implantation.
Imanishi
Toshio
T
Department of Cardiovascular Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama City, Wakayama 641-8510, Japan. t.imani@wakayama-med.ac.jp
Kobayashi
Katsunobu
K
Kuki
Shintaro
S
Takahashi
Chizu
C
Akasaka
Takashi
T
D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000187:drug effects / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000187:drug effects; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020123:Sirolimus / Q000494:pharmacology*; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D022061:Tacrolimus Binding Protein 1A / Q000187:drug effects / Q000235:genetics; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000235:genetics
C436291:CDKN1B protein, human; D007166:Immunosuppressive Agents; D047908:Intracellular Signaling Peptides and Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D012313:RNA; D019098:Telomerase; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus