Differential cyclin E expression in human in-stent stenosis smooth muscle cells identifies targets for selective anti-restenosis therapy.
OBJECTIVE
Cell cycle inhibitors are promising agents to prevent or treat human coronary in-stent stenosis (ISS). However, their lack of specificity for ISS vascular smooth muscle cells (VSMCs) may inhibit medial VSMC proliferation and suppress vessel healing.


METHODS
To identify inhibitor targets that differentially regulate proliferation of ISS vs. medial VSMCs, we examined cell cycle regulation in human VSMCs derived from (A) normal media, (B) ISS sites and (C) primary atherosclerotic plaques (P-VSMCs) using time-lapse videomicroscopy, flow cytometry, immunoblotting and immunohistochemistry.


RESULTS
ISS-VSMC proliferation was intermediate between P-VSMCs and medial VSMCs. Compared with medial cells, P-VSMCs expressed increased p16 and p21, reduced p27, reduced cyclins D(1) and E, and reduced pRb phosphorylation. In contrast, ISS-VSMCs expressed high levels of cyclins E and A with pRb hyperphosphorylation, both in vitro and in vivo, associated with increased and chronic cell proliferation in vivo. Roscovitine, a selective CDK2 inhibitor, inhibited VSMC proliferation by both pRb-dependent and independent pathways and more potently in ISS-VSMCs than medial VSMCs.


CONCLUSIONS
Human ISS-VSMCs have marked differences in the stable expression of multiple cell cycle regulators, suggesting that ISS-VSMCs derive from P-VSMCs driven to proliferate through cyclin E overexpression. The critical role for cyclin E-CDK2 enables the identification of the first agent that selectively inhibits ISS-VSMC proliferation.
O'Sullivan
Michael
M
Division of Cardiovascular Medicine, Cambridge University, Box 110, Addenbrooke's Hospital, CB2 2QQ, Cambridge, UK.
Scott
Stephen D
SD
McCarthy
Nicola
N
Figg
Nicola
N
Shapiro
Leonard M
LM
Kirkpatrick
Peter
P
Bennett
Martin R
MR
D015415:Biomarkers / Q000032:analysis; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D023921:Coronary Stenosis / Q000378:metabolism* / Q000473:pathology; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D009124:Muscle Proteins; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D015607:Stents
D015415:Biomarkers; D019925:Cyclin A; D019927:Cyclin E; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D009124:Muscle Proteins; D011687:Purines; C058992:Tagln protein, mouse; D000077546:Roscovitine; D018844:Cyclin-Dependent Kinases