pathophysiology of atherothrombosis - faid...

Pathophysiology Pathophysiology ofofAtherothrombosisAtherothrombosis

FAOD on Cardiovascular BiomarkersFAOD on Cardiovascular BiomarkersBoston, MABoston, MA October 1, 2007 October 1, 2007

Peter LibbyPeter LibbyBrigham & WomenBrigham & Women!!s Hospitals Hospital

Harvard Medical SchoolHarvard Medical School

Peter Libby, A Fire WithinScientific American May 2002

Peter Libby, A Fire WithinA Fire WithinScientific American May 2002

Inflammation in Atherosclerosis

P. Libby Inflammation in atherosclerosis

Nature 2002;420:868-874.

MacrophageMacrophage

HeterogeneityHeterogeneity

inin

AtherosclerosisAtherosclerosis

Monocyte Monocyte Heterogeneity in MiceHeterogeneity in Mice

Ly-6CLy-6Clolo

Ly-6CLy-6Chihi

Ly-6CLy-6Chihi monocytosis is gradual and systemic monocytosis is gradual and systemic

Ly-6

CL

y-6

Clolo

Mo

no

cyte

sM

on

ocyte

s

Chow Western

ApoE–/–

Ly-

6C

hi

Monocyte

sT

ota

lM

onocyte

s

Ce

lls

/ml

blo

od

Diet (d):

2_106

106

0

2_106

106

0

2_106

106

0

0 3003000

DT (d): 63–86

DT (d): 63–86

DT (d): 59-94

Ce

lls

/ml

blo

od

Ce

lls

/ml

blo

od

Ly-6CLy-6ChihiLy-6CLy-6Clolo

22± 7 81± 6

4 ±2 25±5

CD

90

/B2

20

/DX

5/

NK

1.1

/Ly-6

G

CD11b

Ly-6C

Ev

en

ts

19± 678± 7

Ly-6Clo Ly-6Chi Ly-6Clo Ly-6Chi

Chow Western

ApoE–/–

Ly-6

CL

y-6

Chi

hi

Mo

no

cyte

sM

on

ocyte

s

To

tal

To

tal

Mo

no

cyte

sM

on

ocyte

s

SwirskiSwirski, F. , F. et al.et al. J. J. ClinClin. Invest. 2007;117:195-205. Invest. 2007;117:195-205

DT (d): 41–62

DT (d): 33–38

DT (d): 145-256

Libby P. Inflammation in Atherosclerosis Nature 420:868 (2002)

Mast Cells in Atherosclerosis Mast-cell deficiency mutesMast-cell deficiency mutes

mouse mouse atherogeneisisatherogeneisis

Sun et al., Nature Medicine June 2007

((26 weeks on an 26 weeks on an atherogenic atherogenic diet)diet)

Possible RPossible Rolesoles of Mast Cells in of Mast Cells in AtherothrombosisAtherothrombosis

Peter Libby, A Fire WithinScientific American May 2002

Peter Libby, A Fire WithinA Fire WithinScientific American May 2002

Inflammation

in Atherosclerosis

The traditionalThe traditional

““plumberplumber!!s views view”” of of

the complications ofthe complications of

atherosclerosisatherosclerosis

MIs often arise fromnon-critical stenoses

! Post-thrombolysis

angiography

! Serial angiographic studies

! Intravascular Ultrasound

studiesP Libby

What mechanisms may

cause acute coronarysyndromes in the

absence of criticalstenosis?

Four Mechanisms of Atherosclerotic Plaque DisruptionFour Mechanisms of Atherosclerotic Plaque Disruption

Lib

by

& T

hL

ibb

y &

Th

ééro

ux

, C

irc

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n,

20

05

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x,

Cir

cu

lati

on

, 2

00

5

PlaqueRupture

M.L. Higuchi

ThrombusThrombus

FibrousFibrous

CapCap

Lipid CoreLipid Core

Structural Integrity of the Plaque!s Fibrous Cap

Structural Integrity of the Structural Integrity of the PlaquePlaque!!s Fibrous Cap s Fibrous Cap

•• Depends on interstitialDepends on interstitialcollagen fibrils (types Icollagen fibrils (types I& III) synthesized by& III) synthesized bysmooth muscle cellssmooth muscle cells

Type IType I

CollagenCollagen

GlyGly775775 - - LeuLeu/Ile/Ile776776

Interstitial CollagenasesMMP-1, MMP-8, & MMP-13

3/4 fragment3/4 fragmentCleaved Cleaved

Type IType I

CollagenCollagen

1/4 fragment1/4 fragment

NeoepitopeNeoepitope

Cleaved Collagen Colocalizes with MMP-1 &MMP-13 in Human Atheroma

Cleaved Collagen Colocalizes with MMP-1 &MMP-13 in Human Atheroma

lumen

cleaved type I collagen

MMP-1

lumen

type I collagen

MMP-13

lumen

lumen

GK

Su

kh

ov

a e

t al.

Cir

cu

lati

on

99

:25

03

(1

99

9)

Co

llag

en

ase

Co

llag

en

ase 33

(M

MP

-8)

(M

MP

-8)

Cell T

yp

e

Inflammation and the integrity

of the plaque!s fibrous cap

After Libby P. Circulation 1995

+ + + +

+

+

–

Synthesis Breakdown

Lipid coreLipid core

IL-1IL-1TNF-TNF-""

MCP-1MCP-1M-CSFM-CSF

IFN-IFN-##

CD-40LCD-40L

CollagenaseCollagenaseGelatinasesGelatinases

StromelysinStromelysin

Other proteasesOther proteases

+ + pepidasespepidases

FibrousCapFibrousFibrousCapCap

Collagenase-resistant mutant mice

GLY (775) - ILE (776)

collagenase cleavage site

GLY (775) - PRO (776)

•Mutation at the specific collagenase cleavage

site on type I collagen (“knock-in”)

(Zhao W, Byrne MH, Boyce BF, Krane SM. JCI 1999)

Collagenase-resistance promotes collagenaccumulation in mouse atheroma

Col+/+ / apoE-/- ColR/R / apoE-/-

Pic

ro-s

iriu

s r

ed

sta

inin

g

wit

hp

ola

riza

tio

n

Fukumoto, Deguchi, Libby, Rabkin, Sakata, Chin, Hill, Lawler, Varo, Schoen, Krane, Aikawa

Circulation 2004;110:1953

MmpMmp-13-13+/++/+//apoEapoE-/--/- MmpMmp-13-13-/--/-//apoEapoE-/--/-

MmpMmp-13-13+/++/+//apoEapoE-/--/- MmpMmp-13-13-/--/-//apoEapoE-/--/-

Bright-field illuminationBright-field illumination

Polarized lightPolarized light

MMP-13/MMP-13/collagenasecollagenase-3 deficiency increases-3 deficiency increasesfibrillar fibrillar collagen in mouse collagen in mouse atheromataatheromata

Picrosirius red stainingPicrosirius red staining

5 weeks5 weeks 10 weeks10 weeks

(mm(mm22))

00

0.10.1

0.20.2 Collagen area Collagen area

p<0.05p<0.05

NSNS

MmpMmp-13-13+/+ +/+ MmpMmp-13-13-/--/-

5 weeks5 weeks 10 weeks10 weeks

00

1010

2020(%)(%)

p<0.05p<0.05

Collagen / Collagen / IntimaIntima

NSNS

MmpMmp-13-13+/+ +/+ MmpMmp-13-13-/--/- MmpMmp-13-13+/+ +/+ MmpMmp-13-13-/--/-

MmpMmp-13-13+/+ +/+ MmpMmp-13-13-/--/-

Slide 33

Deguchi Deguchi et al. et al. Circulation 2005; 112:2708Circulation 2005; 112:2708

Plaque rupture with thrombosis

FibrousFibrouscapcap

LipidLipidcorecore

ThrombusThrombusThrombus

FJ Schoen, BWH

Thrombosis on a disruptedatheroma, the cause of most acutecoronary syndromes, results from:

• weakening ofthe fibrous cap

thrombogenicityof the lipid core

CD40 and Tissue Factor in Atheroma

Mach et al. Mach et al. Circulation.Circulation. 1997;96:396. 1997;96:396.

CD40CD40 Tissue FactorTissue Factor

Molecular Biology of theHigh-Risk Plaque


+ + + +

+

+

–

Synthesis Breakdown

Lipid core

IL-1

TNF-"

MCP-1

M-CSF

Fibrous

cap

Fibrous

capIFN-#IFN-IFN-##

CD-40L

Collagen-degrading

Proteinases

Collagen-degradingCollagen-degrading

ProteinasesProteinases

Tissue Factor

Procoagaulant

Tissue FactorTissue Factor

ProcoagaulantProcoagaulant

!!Does the BiologyDoes the Biologyof Atherosclerosisof Atherosclerosis

Suggest UsefulSuggest UsefulBiomarkers?Biomarkers?

Inflammation in AtherosclerosisInflammation in Atherosclerosis

Initiation

Complication

Progression

Libby, Ridker, MaseriCirculation 2002;

105:1135-1143

A New View ofA New View ofVascular DiseasesVascular Diseases

!!Inflammation: a putativeInflammation: a putative““unified theoryunified theory”” of ofatherosclerosisatherosclerosispathogenesispathogenesis

Translation of the basicscience to the clinic:

Translation of the basicscience to the clinic:

!Can we useinflammatory markersto sharpencardiovascular riskprediction?

Circulation

Primary Pro-Inflammatory CytokinesPrimary Pro-Inflammatory Cytokines( e.g., IL-1, TNF-( e.g., IL-1, TNF-"" ))

IL-6IL-6““MessengerMessenger”” Cytokine CytokineICAM-1ICAM-1

SelectinsSelectins, , HSPsHSPs, etc., etc.

LiverLiver

EndotheliumEndotheliumand other cellsand other cells

after Libby, Ridker.Circulation 1999;100:1148–1150.

Pro-Inflammatory PathwaysPro-Inflammatory Pathways

Pro-Inflammatory Risk FactorsPro-Inflammatory Risk Factors

CRPCRPSAASAA

HumanHumanendothelialendothelialcells expresscells expressthe interleukin-the interleukin-11$$ gene geneinduciblyinducibly

Endotoxin and tumornecrosis factor induceinterleukin-1 geneexpression in adulthuman vascularendothelial cellsLibby et al. Am J Path124:179-186 (1986)

Human smoothHuman smoothmuscle cellsmuscle cellsexpress theexpress theTumorTumorNecrosisNecrosisFactor Factor "" gene geneinduciblyinducibly

Warner SJC, Libby P.Warner SJC, Libby P.Human vascular smoothHuman vascular smoothmuscle cells: Target for andmuscle cells: Target for andsource of tumor necrosissource of tumor necrosisfactor. Jfactor. J Immunol Immunol1989;142:100-109.1989;142:100-109.

Circulation




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CRPCRPSAASAA

VCAM-1 expression in rabbit aortaVCAM-1 expression in rabbit aorta

3 weeks on atherogenic diet3 weeks on atherogenic diet3 weeks on atherogenic diet

Li et al. Arteriosclerosis 13:197; 1993Li et al. Arteriosclerosis 13:197; 1993

Circulation




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CRPCRPSAASAA

Proliferating orProliferating orinterleukin-1interleukin-1activated humanactivated humanvascular smoothvascular smoothmuscle cellsmuscle cellssecrete copioussecrete copiousinterleukin-6.interleukin-6.

Loppnow H, Libby P.

J Clin Invest

1990;85:731-738.

S. Kinlay 2002

CD40/ CD40 CD40/ CD40 Ligand Ligand DyadDyad

• Expressed on many cells

including lymphocytes,

macrophages, endothelial,

and VSMC

• Activation increases

expression of CAMs,

Chemokines, Cytokines,

MMPs, Tissue Factor….

• Interruption of the Dyad limits

athero and lipid content in

mice

CD40CD40L (CD154)

Schonbeck U, Libby P. Circ Res 2001; 89:1092-1103

Functional CD 40ligand is expressedon vascularendothelial cells,smooth musclecells, andmacrophages inhuman atheroma

Functional CD 40Functional CD 40ligand ligand is expressedis expressedon vascularon vascularendothelial cells,endothelial cells,smooth musclesmooth musclecells, andcells, andmacrophages inmacrophages inhuman human atheromaatheromaMach F, Schönbeck U,Sukhova GK, Bourcier T,Bonnefoy J-Y, Pober JS, Libby P.PNAS 94:1931 (1997)

Interruption of CD40 signaling inInterruption of CD40 signaling in

hypercholesterolemic hypercholesterolemic mice:mice:

• Inhibits the formation ofatherosclerotic lesions

• Prevents the evolution ofestablished atheroma

• Fosters features of the plaqueassociated with stability

Platelets Release Soluble CD40 Platelets Release Soluble CD40 LigandLigand

• Activated platelets are arichrich source of sCD40L

• Statins may lower sCD40Llevels

CD40L

Andre P, et al. Circulation 2002; 106:896

Schonbeck U, et al. Circulation 2002; 106:2888

Unstimulated

Platelets

Stimulated

Platelets

S. Kinlay 2002 S. Kinlay 2002

sCD40L

Inflammation Biology of theHigh-Risk Plaque


+ + + +

+

+

–

Synthesis Breakdown

Lipid core

IL-1

TNF-"

MCP-1

M-CSF

Fibrous

cap

Fibrous

capIFN-#IFN-IFN-##

CD-40L

Matrix-Degrading

Proteinases

Matrix-DegradingMatrix-Degrading


Tissue Factor

Procoagulant

Tissue FactorTissue Factor

ProcoagulantProcoagulant

Matrix Metalloproteinase-9 inHuman Atheroma

Galis e

t al. J

Clin

In

vest

94:2

493 (

1994)

Matrixmetallo-

proteinaseactivity

in extractsof humanatheroma

J Clin Invest94:2493 (1994)

Plasma concentrations andPlasma concentrations andgenetic variation of matrixgenetic variation of matrix

metalloproteinase 9 andmetalloproteinase 9 andprognosis of patients withprognosis of patients with

cardiovascular diseasecardiovascular disease

BlankenbergBlankenberg, , RupprechtRupprecht, Poirier, Bickel,, Poirier, Bickel,SmiejaSmieja, , HafnerHafner, Meyer, , Meyer, CambienCambien, , TiretTiret;;

AtheroGene AtheroGene Investigators.Investigators.

Circulation. 2003;107:1579-85.Circulation. 2003;107:1579-85.Plasma concentrations and genetic variation of matrix metalloproteinase 9Plasma concentrations and genetic variation of matrix metalloproteinase 9and prognosis of patients with cardiovascular disease. and prognosis of patients with cardiovascular disease. Blankenberg Blankenberg et al.et al.

AtheroGene AtheroGene Investigators. Circulation. 2003;107:1579-85.Investigators. Circulation. 2003;107:1579-85.

1127 CAD Pts

Inflammation Biology of theHigh-Risk Plaque


+ + + +

+

+

–

Synthesis Breakdown

Lipid core

IL-1

TNF-"

MCP-1

M-CSF

Fibrous

cap

Fibrous

capIFN-#IFN-IFN-##

CD-40L

Collagen-degrading

Proteinases

Collagen-degradingCollagen-degrading


Myeloperoxidase

Superoxide

Peroxynitrite

MyeloperoxidaseMyeloperoxidase

SuperoxideSuperoxide

PeroxynitritePeroxynitrite

MyeloperoxidaseMyeloperoxidase!An enzyme released by activated

granulocytes and a monocytesubpopulation

!Bound to extracellular matrix atsites of inflammation

!!ConvertsConverts Cl- + H2O2 to HOCl(hypochlorous acid), an oxidant andchlorinating species

Macrophage Macrophage myeloperoxidase myeloperoxidase regulation inregulation inhuman atherosclerosis: implications forhuman atherosclerosis: implications for

acute coronary syndromesacute coronary syndromes

Sugiyama et al. Am J Pathol 2001; 158:879-891

Inflammation and Thrombosis

Platelet-derived growth factor

Platelet factor 4

CD 154 (CD40L)

RANTES (PMN chemokine)

Thrombospondin

Transforming growth factor-$

Nitric oxide

Activated platelets produce inflammatory modulators

Libby & Simon. Libby & Simon. CirculationCirculation 2001 2001

Croce &, Libby Croce &, Libby Curr Opin HematolCurr Opin Hematol. . 20072007

Study ObjectivesStudy Objectives

•• To identify genes responsible for acuteTo identify genes responsible for acute

thrombotic complications of CADthrombotic complications of CAD

(transcriptional profiling of STEMI vs.(transcriptional profiling of STEMI vs.

CAD)CAD)

•• To develop novel biomarkers forTo develop novel biomarkers for

cardiovascular disease and newcardiovascular disease and new

targets for therapy of atherosclerosistargets for therapy of atherosclerosis

Transcriptional profiling:

The causality problem

Gene expression following acute MI may

reflect either triggering events or

downstream consequences of plaque

rupture and thrombosis, thereby precluding

definitive conclusions regarding causality.

•• Since platelets lackSince platelets lack

nuclear DNA, butnuclear DNA, but

retain megakaryocyte-retain megakaryocyte-

derived mRNAsderived mRNAs, the, the

platelet transcriptomeplatelet transcriptome

provides a novelprovides a novel

window on genewindow on gene

expression expression precedingprecedingacute coronary eventsacute coronary events..

The platelet The platelet transcriptometranscriptome

provides a snapshot in timeprovides a snapshot in time

Platelets circulatefor ~ 7 days

Cardiac Catheterization Laboratory

Brigham and Women’s Hospital

Cardiac Catheterization LaboratoryCardiac Catheterization Laboratory

Brigham and WomenBrigham and Women’’s Hospitals Hospital

RNA isolated from platelet-rich plasma from

anticoagulated whole blood (up to 50-100

ng/50 mL), amplified, and profiled

RNA isolated from platelet-rich plasma fromRNA isolated from platelet-rich plasma from

anticoagulated anticoagulated whole blood (up to 50-100whole blood (up to 50-100

ng/50 ng/50 mLmL), amplified, and profiled), amplified, and profiled

Normal(no CAD)

NormalNormal

(no CAD)(no CAD)StableAngina

StableStable

AnginaAnginaUnstableAngina

UnstableUnstable

AnginaAnginaSTEMISTEMISTEMI

Patients with ST-segment-elevationPatients with ST-segment-elevation

myocardial myocardial infacrction infacrction (STEMI) have higher(STEMI) have higher

plasma levels of MRP-14 than stable patientsplasma levels of MRP-14 than stable patients

• In humans, MRP-14 exists primarily as the

heterodimer MRP-8/14.

• Neutrophil MRP-8/14 is secreted in response to

inflammation and accumulates in plasma.

• MRP-8/14 levels were increased in the plasma

of patients presenting with STEMI vs. CAD,

(median 17 mg/mL, IQR 14.8, 19.7) vs. (8.0

mg/mL, IQR 5.0, 10.6;Healy et al. Circulation 2006;113:2278-2284

Validation Phase:

Women’s Health Study

•• Prospective, nested case-control studyProspective, nested case-control study

(255 case-control pairs) in population of(255 case-control pairs) in population of

apparently healthy post-menopausalapparently healthy post-menopausal

women to assess the risk of firstwomen to assess the risk of first

cardiovascular event (CV death, non-fatalcardiovascular event (CV death, non-fatal

MI, stroke) associated with base-lineMI, stroke) associated with base-line

plasma levels of MRP-8/14.plasma levels of MRP-8/14.Ridker, PM et. al. NEJM 2005

MRP-8/14 improvedMRP-8/14 improved

risk-prediction modelsrisk-prediction models

based on totalbased on total

cholesterol:HDL orcholesterol:HDL or

CRP testing alone.CRP testing alone.

MRP-8/14MRP-8/14

independentlyindependently

predicts CV risk inpredicts CV risk in

the the WomensWomens’’ HeathHeath

StudyStudy

Healy et al. Circulation 2006;113:2278-2284

Circulation




LiverLiver





CRPCRPSAASAA

The dominant emergingThe dominant emergingbiomarker of cardiovascular riskbiomarker of cardiovascular risk

C-reactive protein: CRP

pathophysiology of atherothrombosis - faid...

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