Drug Eluting Stents – The Cell Biology Andrew Newby BRISTOL HEART INSTITUTE ?

Basis of neointima formation ONE WEEK Thrombus Inflammation Injury Stretch

Early thrombus and later inflammation % of specimens Thrombus Neutrophils Macrophages Giant cells <3 d Histological findings in 55 coronary in-stent restenosis specimens Farb et. al. Circulation 1999;95: d d >30 d

More injury - more restenosis Serial IVUS - Hoffman et. al. Am J Cardiol 1999;83:1170-4

Smooth muscle cells and extracellular matrix % of specimens Smooth muscle cells Proteoglycans and HA <3 d d d 45 >30 d 100 Farb et. al. Circulation 1999;95:44-52 SMC are 59% of all cells 25% of SMC PCNA, cyclinE, and cdk2 positive Kearney et. al. Circulation 1997;95:

Where do neointimal cells come from? Adventitial fibroblasts? Medial SMC? Circulating stem cells?

Importance of medial injury Plaque Intact media Damaged media Intimal thickness (mm) Farb et. al. Circulation 1999 ;95:44-52

Growth factors promote neointima formation (Russell Ross) mm SMC PDGF SMC bFGF IGF-1, TGF  Thrombin, 5HT PDGF mm

SMC in intact aorta don’t respond to growth factor – FCS induced thymidine incorporation T Izzard et. al. Cardiovasc Res 2002: 53;

Control of smooth muscle cell cycle G1 S G2Mitosis G0 Growth factors Signal Trans- duction MAPK CyclinD/ cdk4 Decreased P 16 and p 27 ckis CyclinE/ cdk2 R cdk cyclin T Y P CKI

p27 levels are maintained and cdk4 kinase activity is inhibited in intact vessels Intact Isolated Intact Isolated h0.5 p27 CKI h CyclinD/cdk4 activity T Izzard et. al. Cardiovasc Res 2002: 53;

Dual control of smooth muscle cell proliferation G1 S G2Mitosis G0 Growth factors Signal Trans- duction MAPK CyclinD/ cdk4 Decreased P 16 and p 27 ckis CyclinE/ cdk2 R Cell Cycle Matrix protein binding to cell surface integrins

Basis of SMC migration Matrix proteins Signal Transduction Pathways Activation of CAM- kinase II Engagement of cell surface integrins Cytoskel- etal re- arrangement Chemo- attractant Migration

Matrix regulation of SMC Contractile, quiescent SMC BM, laminin Type IV collagen Fibronectin Synthetic, migrating proliferating SMC Fibronectin, osteopontin, vitronectin, Type I, IV, VIII collagen, Versican, hyaluronic acid

Extracellular proteases combine with growth factors to promote neointima mm SMC MMPs SMC MMPs, uPA, other proteases Migrate Divide mm

‘Multiple key control’ of the cell cycle G1 4,5PIP2 – 3,4,5 PIP3 G0 PDGF PI3K Grb SOS raf PKBmTOR PLC  I1,4,5P3 DAG PKC rasMEKERK1/2 PROTEIN KINASES

Steps regulated by mammalian target of rapamycin G1S Migration! PDGF PI3K PKB mTOR p27 CKI Matrix proteins EIF-4E Protein synthesis p70 s6k Gene expression Marx and Marks, Circulation 2001;104: Sun, Circulation 2001;103:

Inhibition of mTOR by sacrolimus (rapamycin) not tacrolimus G1S Migration! mTOR p27 CKI EIF-4E Protein synthesis p70 s6k Gene expression FKB Sirolimus Tacrolimus Suzuki et al. Circulation 2001;104: Chemokines MCP-1, IL-6 CN

Microtubules mediate migration and mitosis InterphaseMitosis

Paclitaxel disrupts the cytoskeleton and inhibits SMC migration and proliferation Axel et al Circulation 1997; 96: actin vimentin

Conclusions –inhibition of SMC proliferation G1 S G2Mitosis G0 Rapamycin - antiproliferative Signal Trans- duction MAPK CyclinD/ cdk4 Decreased P 16 and p 27 ckis CyclinE/ cdk2 R Cell Cycle Matrix proteins MMPI Taxol – antimitotic

Conclusions –inhibition of SMC migration Rapamycin Decreased p 27 cki Matrix proteins MMPI Signal Transduction Pathways Activation of CAM- kinase II Engagement of cell surface integrins Chemo- attractant Taxol Cytoskel- etal rear- rangement

Rapamycin The jury considers the evidence