Rapamycin inhibits human in stent restenosis vascular smooth muscle cells independently of pRB phosphorylation and p53.
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 G(1)-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 G(1), as cell proliferation (but not cell size) of S/G(2)-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 p70(S6K), 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 G(1). Rapamycin's action does not require p53 or pRB. We show that p70(S6K) 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.
Rosner
Dalya
D
Unit of Cardiovascular Medicine, University of Cambridge Clinical School of Medicine, Addenbrooke's Centre for Clinical Investigation, Addenbrooke's Hospital, UK.
McCarthy
Nicola
N
Bennett
Martin
M
D050256:Antimitotic Agents / Q000627:therapeutic use*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D023903:Coronary Restenosis / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D003864:Depression, Chemical; D006801:Humans; D007399:Interphase; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D020123:Sirolimus / Q000627:therapeutic use*; D015607:Stents; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D050256:Antimitotic Agents; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus