Drug Eluting Stents – The Cell Biology

Andrew Newby BRISTOL HEART

INSTITUTE

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Basis of neointima formation

ONE WEEK

Thrombus

Inflammation

Injury

Stretch

Early thrombus and later inflammation

% of specimens•Thrombus•Neutrophils•Macrophages•Giant cells

<3 d7279820

Histological findings in 55 coronary in-stent restenosis

specimens Farb et. al. Circulation 1999;95:44-52

4-11 d7883670

12-30 d24729710

>30 d008529

More injury - more restenosis

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

Smooth muscle cells and extracellular matrix

% of specimensSmooth muscle cellsProteoglycans and HA

<3 d0

4-11 d0

12-30 d45

>30 d100

Farb et. al. Circulation 1999;95:44-52

SMC are 59% of all cells25% of SMC PCNA, cyclinE, and cdk2 positive

Kearney et. al. Circulation 1997;95:1998-2002

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)

.2

.4

.6

.8

1

0

Farb et. al. Circulation 1999;95:44-52

Growth factors promote neointima formation (Russell Ross)

m

SMC

SMC

SMC

PDGF

SMC

SMC

SMCSMC

SMC

bFGFIGF-1, TGF

Thrombin, 5HT

PDGF

m

SMC in intact aorta don’t respond to growth factor –FCS induced thymidine incorporation

0.00E+00

2.00E+06

4.00E+06

6.00E+06

0 10 20 30 40 50 hours

DP

M/

g D

NA

isolated cells

aortic tissue

T Izzard et. al. Cardiovasc Res2002: 53; 242-52

Control of smooth muscle cell cycle

G1

S

G2Mitosis

G0Growthfactors

SignalTrans-ductionMAPK

CyclinD/cdk4

DecreasedP16 and p27

ckis

CyclinE/cdk2

R

cdk

cyclinT

Y P

CK

I

p27 levels are maintained and cdk4 kinase activity is inhibited in intact vessels

Intact

Isolated

Intact

Isolated

0 1 2 4 6 8 12 16 24 h0.5

p27 CKI

0 4 8 12 16 24 h

CyclinD/cdk4 activity T Izzard et. al. Cardiovasc Res2002: 53; 242-52

Dual control of smooth muscle cell proliferation

G1

S

G2Mitosis

G0Growthfactors

SignalTrans-ductionMAPK

CyclinD/cdk4

DecreasedP16 and p27

ckis

CyclinE/cdk2

R

Cell Cycle

Matrix protein binding to cell surface integrins

Basis of SMC migration

Matrix proteins

SignalTransduction

Pathways

Activationof CAM-kinase II

Engagementof cell

surfaceintegrins

Cytoskel-etal re-

arrangementChemo-attractant

Migration

Matrix regulation of SMC

Contractile, quiescent SMC

BM, lamininType IV collagen

Fibronectin

Synthetic, migratingproliferating SMC

Fibronectin, osteopontin, vitronectin,Type I, IV, VIII collagen,Versican, hyaluronic acid

Extracellular proteases combine with growth factors to promote neointima

m

SMC

SMC

SMC

MMPs

SMC

SMC

SMCSMC

SMC

MMPs, uPA, other proteases

Migrate

Divide

m

‘Multiple key control’ of the cell cycle

G1

4,5PIP2 – 3,4,5 PIP3G0

PDGF

PI3K

Grb

SOS

raf

PKB mTOR

PLCI1,4,5P3DAG

PKC

ras MEK ERK1/2

PROTEIN KINASES

Steps regulated by mammalian target of rapamycin

G1SMigration!PDGF

PI3K PKB

mTOR

p27 CKI

Matrix proteins

EIF-4EProtein synthesis

p70s6k

Gene expression

Marx and Marks, Circulation 2001;104:852-5. Sun, Circulation 2001;103:2967-72

Inhibition of mTOR by sacrolimus (rapamycin) not tacrolimus

G1SMigration!

mTOR

p27 CKI

EIF-4EProtein synthesis

p70s6k

Gene expression

FKB

FKB

Sirolimus

Tacrolimus

Suzuki et al. Circulation 2001;104:1188-93

ChemokinesMCP-1, IL-6

CN

Microtubules mediate migration and mitosis

Interphase Mitosis

Paclitaxel disrupts the cytoskeleton and inhibits SMC migration and proliferation

Axel et al Circulation 1997; 96:636-45

actin

vimentin

Conclusions –inhibition of SMC proliferation

G1

S

G2Mitosis

G0

Rapamycin -antiproliferative

SignalTrans-ductionMAPK

CyclinD/cdk4

DecreasedP16 and p27

ckis

CyclinE/cdk2

R

Cell Cycle

Matrix proteins

MMPI

Taxol –antimitotic

Conclusions –inhibition of SMC migration

Rapamycin

Decreasedp27

cki

Matrix proteins

MMPI

SignalTransduction

Pathways

Activationof CAM-kinase II

Engagementof cell

surfaceintegrins

Chemo-attractant

Taxol

Cytoskel-etal rear-

rangement

RapamycinThe jury considers the evidence