Copyright © 2005, European Society of Cardiology
Rapamycin inhibits human in stent restenosis vascular smooth muscle cells independently of pRB phosphorylation and p53
Unit of Cardiovascular Medicine, University of Cambridge Clinical School of Medicine, Addenbrooke's Centre for Clinical Investigation, Box 110, Addenbrooke's Hospital, Cambridge, CB2 2QQ, UK
* Corresponding author. Tel.: +44 1223 331504; fax: +44 1223 331505. Email address: mrb{at}mole.bio.cam.ac.uk
Objective: Drug-eluting stents containing the immunosuppressant rapamycin markedly inhibit in stent restenosis (ISR). However, the molecular mechanisms that underlie its effect on ISR-derived vascular smooth muscle cells (VSMCs), as opposed to normal VSMCs, are unknown. Specifically, as ISR-VSMCs have altered cell cycle regulation, rapamycin may arrest these cells via novel molecular pathways.
Methods: We isolated human VSMCs from sites of ISR, and examined the effect of rapamycin on cell proliferation using MTT assay, time lapse videomicroscopy and flow cytometry. Regulation of G1–S transition was examined using Western blotting, and cell size and protein synthesis examined using flow cytometry and collagen assay, respectively. The requirement for pRB and p53 was examined using ISR VSMCs expressing E1A and a dominant negative p53, respectively.
Results: ISR-VSMC proliferation was potently inhibited by rapamycin. Arrest was confined to G1, as cell proliferation (but not cell size) of S/G2-arrested cells was unaffected by rapamycin. Moreover, ISR-VSMC lines generated with disrupted p53 or pRB function still arrested in the presence of rapamycin, suggesting that these genes are dispensable for rapamycin-induced arrest. Significantly, rapamycin completely inhibited the phosphorylation of p70S6K, an mTOR-regulated kinase implicated in the control of proliferation, but had no effect on collagen or total protein synthesis.
Conclusions: We demonstrate that rapamycin is a potent inhibitor of ISR VSMC proliferation during G1. Rapamycin's action does not require p53 or pRB. We show that p70S6K is markedly inhibited in rapamycin-arrested ISR cells, suggesting that regulation of its upstream kinase, mTOR, is important for the control of proliferation in ISR cells.
KEYWORDS Restenosis; Stents; Smooth muscle; Atherosclerosis
Time for primary review 23 days
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