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PRE-CLINICAL RESEARCH

Sirolimus and Everolimus Induce Endothelial Cellular Senescence Via Sirtuin 1 Down-Regulation

Therapeutic Implication of Cilostazol After Drug-Eluting Stent Implantation

Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Manuscript received October 19, 2008; revised manuscript received December 17, 2008, accepted January 27, 2009.

^_* Reprint requests and correspondence: Dr. Yasuyoshi Ouchi, Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan (Email: youchi-tky{at}umin.ac.jp).

Objectives: The aim of this study was to compare the effects of paclitaxel, sirolimus, and everolimus on the senescent phenotype in human endothelial cells, and to further investigate possible involvement of mammalian sirtuin 1 (Sirt1) down-regulation as a mechanism.

Background: Endothelial cell senescence may play a role in impaired re-endothelialization after drug-eluting stent (DES) implantation. Recently, the down-regulation of Sirt1 has been shown to mediate oxidative stress-induced endothelial senescence.

Methods: Senescent human umbilical vein endothelial cells (HUVEC) were judged by senescence-associated β-galactosidase assay (SA-βgal), morphological appearance, and plasminogen activator inhibitor (PAI)-1.

Results: Treatment with paclitaxel, sirolimus, and everolimus significantly caused a senescent phenotype and PAI-1 up-regulation, associated with a decrease in endothelial nitric oxide synthase (eNOS) and Sirt1 expression. Overexpression of Sirt1 or Sirt1 activation reversed the sirolimus- or everolimus-induced senescent phenotype. Interestingly, paclitaxel-induced senescence was not suppressed by Sirt1 overexpression, suggesting the existence of a different mechanism. Cilostazol markedly inhibited the sirolimus- or everolimus-induced senescent phenotype (sirolimus or everolimus [2.5 nmol/l]; 49.2% or 53.0% SA-βgal positive vs. only 13.6% or 14.6% with cilostazol [100 µmol/l]) and PAI-1 up-regulation, but had no influence on the effects of paclitaxel. Finally, aspirin significantly blunted sirolimus- or everolimus-induced senescence, but neither ticlopidine nor clopidogrel had any effects.

Conclusions: Sirolimus and everolimus induce endothelial senescence involving down-regulation of Sirt1. In contrast, the development of endothelial senescence by paclitaxel involves a Sirt1-independent pathway. Because sirolimus and everolimus are involved in Sirt1 modulation, cilostazol rescues HUVEC from sirolimus- or everolimus-induced senescence. These results may have therapeutic implications in the clinical sequelae after DES implantation.

Key Words: sirolimus • everolimus • antiplatelet therapy • endothelial senescence

Abbreviations and Acronyms   DES = drug-eluting stent(s)   EGM = endothelial growth medium   eNOS = endothelial nitric oxide synthase   HUVEC = human umbilical vein endothelial cells   PAI = plasminogen activator inhibitor   SA-βgal = senescence-associated β-galactosidase assay   SES = sirolimus-eluting stent(s)   Sirt1 = silent mating type information regulation 2 homolog 1   ST = stent thrombosis

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