Characteristics of Vulnerable Characteristics of Vulnerable Plaque: Structural Observations Plaque: Structural Observations and Natural History Insights from and Natural History Insights from

the Core Pathology Laboratorythe Core Pathology Laboratory

2323rdrd AprilApril, , 20082008

Renu Virmani, MDCVPath Institute Inc., 19 Firstfield Road,

Gaithersburg,Maryland, USA

Conflict of Interest StatementWithin the past 12 months, I or my spouse/partner have had a financial interest/arrangement or affiliation with the organization(s) listed below.

Physician Name: Renu Virmani, M.D.

Company/Relationship:Research GrantsMedtronic AVE; Guidant; Abbott; GE Healthcare; Takeda; Atrium Medical Corp.; ev3; Conor Medsystems; TopSpin Medical (Israel) Ltd.; Paracor Medical, Inc.; OrbusNeich; Terumo Corp.; Vascular Therapies, LLC; CardioKinetix; Osiris Therapeutics, Inc.; Edwards Life Sciences; Biomerix; Nitinol Device and Components; Sorin Biomedica Cardio S.r.l; 3F Therapeutics; Hancock Jaffee Labs, Inc.; Cardiovascular Device Design; Coaptus; Biotegra; Cardica, Inc.; Cordis Corp.; Cryo Vascular Systems, Inc.; CVRx, Inc.; diaDexus, Inc.; InfraReDx, Inc.; Kensey Nash Corp.; Medeikon Corp.; MedNova USA, Inc.; Microvention, Inc.; Oregon Medical Laser Center; Spectranetics Corp.; Takeda Pharmaceuticals North America; Toray Industries, Inc.; Vascular Concepts; Volcano Therapeutics, Inc.; BioSensors International; Alchimer S.A. and Relisys.

Consultant: Medtronic AVE; Guidant; W.L. Gore; CryoVascular Systems, Inc.; Volcano Therapeutics Inc.; Precient Medical; Medeikon; CardioMind, Inc.; Direct Flow; and Atrium Medical Corp.

Employment 25%: Cardiovascular Research FoundationDo not own any stock in any company.

Within the past 12 months, I or my spouse/partner have had a financial interest/arrangement or affiliation with the organization(s) listed below.

Physician Name: Renu Virmani, M.D.

Company/Relationship:Research GrantsMedtronic AVE; Guidant; Abbott; GE Healthcare; Takeda; Atrium Medical Corp.; ev3; Conor Medsystems; TopSpin Medical (Israel) Ltd.; Paracor Medical, Inc.; OrbusNeich; Terumo Corp.; Vascular Therapies, LLC; CardioKinetix; Osiris Therapeutics, Inc.; Edwards Life Sciences; Biomerix; Nitinol Device and Components; Sorin Biomedica Cardio S.r.l; 3F Therapeutics; Hancock Jaffee Labs, Inc.; Cardiovascular Device Design; Coaptus; Biotegra; Cardica, Inc.; Cordis Corp.; Cryo Vascular Systems, Inc.; CVRx, Inc.; diaDexus, Inc.; InfraReDx, Inc.; Kensey Nash Corp.; Medeikon Corp.; MedNova USA, Inc.; Microvention, Inc.; Oregon Medical Laser Center; Spectranetics Corp.; Takeda Pharmaceuticals North America; Toray Industries, Inc.; Vascular Concepts; Volcano Therapeutics, Inc.; BioSensors International; Alchimer S.A. and Relisys.

Consultant: Medtronic AVE; Guidant; W.L. Gore; CryoVascular Systems, Inc.; Volcano Therapeutics Inc.; Precient Medical; Medeikon; CardioMind, Inc.; Direct Flow; and Atrium Medical Corp.

Employment 25%: Cardiovascular Research FoundationDo not own any stock in any company.

Natural History of Atherosclerosis

• Systemic factors - hyperlipidemia, diabetes mellitus, smoking, hypertension, age and sex, hsCRP, Lp-PLA2, etc.

• Local factors: at branch points, e.g., carotid bifurcation, abdominal aorta just above bifurcation coronary branch point, and arch vessels at take off, are the sites of atherosclerosis manifestation

• Thrombosis occurs in the coronary arteries at focal points and is most often seen in the proximal segments of the three main coronary arteries (systemic coagulation factors play a role), and occur at sites where there are underlying plaque characteristic that result in thrombosis

Branch points are the sites of atherosclerosis and occur in areas of low shear

A

Carotid ArteryLeft Coronary arteryC

Low shear

High shearBlood flow

Lesions with ThrombiLesions with Thrombi

• Plaque Rupture• Plaque Erosion• Calcified Nodule

Causes of Coronary ThrombosisCauses of Coronary Thrombosis

Calcified noduleCalcified noduleErosionErosion

ThTh NC

CalcifiedNodule

FC

RuptureRupture

Th

RuptureSiteNC

Th

Th

Th Th Th

Virmani R, et al. Arterioscler Thromb Vasc Biol 2000;20:1262

MΦ

T cells SMCs

HLA-DR

ThTh

NC

A

B

Th

NC Gross and Light MicroscopicFeatures of Plaque Rupture

60% of Thrombi in Sudden Coronary Death occur form Plaque Rupture

E

D

G

F

C

Th

NC

Fig 3-1

Plaque Erosion: 30-35% of thrombi in SCDPlaque erosion in a 33 year-old female complaining of chest pain for two-weeks and discharged from the emergencyroom with a diagnoses of anxiety.

A

E

C

D FMΦ

T-cells PLT Fibrin

NC

B

SMCs

Necrotic core

Th

Lumen

Th

Lumen

Plaque rupture Plaque erosion Calcified noduleCalcified nodule

Clinical and Morphologic Difference in Plaques Associated Clinical and Morphologic Difference in Plaques Associated with Luminal Thrombiwith Luminal Thrombi

ThTh

60% thrombi in SCDM>F, Older, Ca++

Eccentric = concentricGreater % stenosisMacs, T cells,HLADr

30-35% thrombi in SCDM=F, youngerUsually eccentricLesser % stenosisSMC rich, proteoglycans

2-7% thrombi in SCD, calcified platesM>F, older, mid RCAUsually eccentricStenosis variableNodules of bone

Acute Coronary SyndromeAcute Coronary Syndrome• Acute Myocardial Infarction

– >90% have coronary thrombi, usually occlusive– 65-75% from plaque rupture– 25-35% from plaque erosion (Arbustini E, et al. Heart 1999)

• Unstable Angina Pectoris– 70% have thrombi, mostly non-occulsive (Mizuno K, et al. Lancet

1991. Ueda Y et al. JACC 1996) – Distribution weather rupture or erosion, unknown

• Sudden Coronary Death– 60% have thrombi- 60% rupture, 35% erosion, 2-5% calcified

nodule)– 40% have stable plaques with >75% x-sectional narrowing (no

thrombus)– 40% have HMI and 15% AMI (Virmani R, et al. ATVB 2000

Development of Human Coronary AtherosclerosisPathologicintimal thickening

Fibrouscap atheroma

Thin-capFibroatheroma

IntimalIntimalthickening

IntimalIntimalxanthomathickening xanthoma

LP NCNC

FC

Extracellular lipid

Necrotic coreCholesterol clefts

Calcified plaque

Healed thrombus

Macrophage foam cells HemorrhageThrombus

Smooth muscle cells

Collagen

FC = fibrous capLP = lipid pool NC = necrotic core

Thin-Cap Atheroma (Vulnerable Plaque) Components

• Necrotic core• Thin fibrous cap (< 65 µm)• Cap infiltrated by macrophages and

lymphocytes• Cap composition – type 1 collagen and

few smooth muscle cells

Thin cap Fibroatheroma (Vulnerable Plaque)

AA AB

Movat

nc

nc

Fibrous cap CD68

nc

AC

AE AF

vWFCD68

AD

Morphologic Characteristics of Plaque Rupture and Thin-cap Fibroatheromas

NecroticCore (%)

Fibrous capThickness (µm)

SMCs(%)

T-lymph

CalcificationScore

MΦs(%)Plaque type

Rupture

Thin-capFibroatheroma

P value

34±17

23±17

0.01

23±19

<65µm

26±20

14±10

0.005

0.002±0.004

6.6±10.4

ns

4.9±4.3

6.6±10.4

ns

1.53±1.03

0.97±1.1

0.014

Mean values are represented ± standard deviation. Abbreviations: MΦs= macrophages,SMCs= smooth muscle cells, T-lymph= T-lymphocytes

Kolodgie F, et al. Current Opinion in Cardiology 2001;16:285

Relationship of Fibrous Cap Thickness to Macrophage Infiltration

0

1

2

3

4

5

6

Cell Mean for % Kp-1

P = 0.03P = 0.06

Less

than

65

µm

66 -

200

µm

201

-300

µm

Mor

e th

an 3

00 µ

m

Remodeling in Varying CoronaryLesion Morphologies

B.A.

IEL-

Expe

cted

IEL

(mm

2

3

)

Eros

ion

Stab

le

Thin

cap

ath

erom

a

Plaq

ue h

emor

rhag

e

Acu

te ru

ptur

e

Hea

led

rupt

ure

Tota

l occ

lusi

on

IEL-

Expe

cted

IEL

(/pla

que

area

)

54321

2

1

0

-1

Eros

ion

Stab

le

Thin

cap

ath

erom

a

Plaq

ue h

emor

rhag

e

Acu

te ru

ptur

e

Hea

led

rupt

ure

Tota

l occ

lusi

on

0-1-2-3

Plaque rupture with mild non Plaque rupture with mild non occlusive thrombus: occlusive thrombus: mechanism by which mechanism by which

plaques progressplaques progress

2829389

Proximal

Rupture site

th

th

th

th

3mm6mm

9mm

Do TCFAs lead to plaque progression ?Do TCFAs lead to plaque progression ?Movat pentachrome

Sirius red Sirius red with polarized lightBurke AP, et al. Circulation 2001

Mean % stenosis increases with number of prior rupture sites

0

10

20

30

40

50

60

70

80

90

1 2 3 40

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4

Number of prior ruptures, acute rupture sites

Mean

% s

teno

sis

B

Mean

% s

teno

sis

ANumber of prior ruptures, healed rupture sites

Burke, A P et al. Circulation 2001;103:9364-940

Thin-cap FibroatheromaRecent Intraplaque Hemorrhage is seen at

Multiple sites in Patients Dying SCD

A

a

B

b

00.51

1.52

2.53

3.54

4.55

Plaquerupture

Plaqueerosion

Severe CAD>75%

Plaque Hemorrhage

Virmani R, et al. ATVB 2005

Consequence of Extravasated Erythrocytes Outside the

Vasculature• Free cholesterol content of erythrocyte membrane

exceeds that of all other cells in the body, with lipid constituting 40% of the weight

• Yeagle in 1985 showed that extravasated erythrocytes contain free cholesterol and Arbustini et al. in 2002 showed macrophage infiltration in intimal plaques in pulmonary trunk of patients with pulmonary hypertension at sites containing erythrocyte membranes

We examined tissues from nonvascular location to determine the effect of hemorrhage

• Pericardial hemorrhage• Intratumor hemorrhage (atrial hemangiomas, papillary

carcinoma of kidney etc)Kolodgie FD, et al. New Engl J Med 2003

E

H

Thin Fibrous Cap Atheroma

Phase Separation of Erythrocyte-Derived CholesteroIin Coronary and Non-Coronary Diseases

Hemorrhagic Pericarditis

F

E

HemorrhagePeriphery

HemorrhageCore

RBCs

MΦ

C

A

BD GpA

MΦ

GpA

Plaque Types Studied Plaque Types Studied A. B.Pathologic Intima Thickening

Fibroatheroma ‘Late’ Core

CD68

NC

Fibroatheroma ‘Early’ Core

LP

CD68

NC

CD68

C.Thin Cap FibroatheromaD.

NC

CD68

Morphometric Analysis of Hemorrhagic Events in Human Morphometric Analysis of Hemorrhagic Events in Human Coronary Plaques from Sudden Death VictimsCoronary Plaques from Sudden Death Victims

Necrotic Core(mm2)

GpAScore

MΦ(mm2)IronPlaque Type

PIT no core(n=129)FA early core(n=79)FA late core(n=105)TCFA(n=52)

0.09±0.04

0.23±0.07

*0.94±0.11

*1.60±0.20

0.07±0.05

0.17±0.08

*0.41±0.09

*1.24±0.24

0.0

0.06±0.02

*0.84±0.08

*1.95±0.30

0.002±0.001

0.018±0.004

*0.059±0.007

*0.142±0.016

Values are reported as the means±SE, *p<0.001 versus early core. The number in parenthesis represent thenumber of lesions examined;the total number= 365. MΦ = macrophages

Kolodgie FD, et al. New Engl J Med 2003

Plaque Vasa VasorumPlaque Vasa Vasorum• Plaque capillaries are observed in atherosclerotic

plaques with plaque thickness > 0.5 mm, suggesting that wall ischemia may be a determinant of neovascularization.

• Heistead and Armstrong reported a 5 fold increase in intimal/medial blood flow from proliferating micro vessels in monkeys fed a high cholesterol diet for 17 months. (Arteriosclerosis 1986)

• Plaque Vv may be a potential source of inflammation within the plaque [expression of VCAM-1, ICAM-1 and E-selectin has been shown in plaque Vv (O’Brian, et al. AJP 1994).

• Inflammation and matrix composition of atherosclerotic plaques may also influence angiogenesis.

Virmani R, et al., Arteriosclero Throm Vac Biol 2005;20:1262

Adventitial Vasa Vasorum Adventitial Vasa Vasorum Heterogeneity among different Heterogeneity among different

vascular bedsvascular beds

0

0.5

1

1.5

2

2.5

3

Coronary Renal Carotid Femoral

Vasa vasorumdensity/mm2

P<0.05P<0.05

P<0.05

P<0.05

Adv

entit

ial v

asa

vaso

rum

/mm

2

Low vasa vasorum density in internal thoracic artery may be responsible for the low incidence of atherosclerosis

Gallili et al. J Vasc Surg 2004;40:529 and J Thorac Cardiovasc Surg 2005;129:767

Vasa vasorum at various stages of

plaque development

Adaptive Intimal thickeningVv in adventitia

Fibroatherma Abnormal Vv Fibroatherma with leaky Vv

Intraplaque Vasa Vasorum in Coronary Intraplaque Vasa Vasorum in Coronary Plaques with a Necrotic CorePlaques with a Necrotic Core

A B150 µm thick sections stained with Ulex

nc nc

Virmani R, et al., Arteriosclero Throm Vac Biol 2005;20:1262

Vasa Vasorum by Plaque Type K Mean No. of Vasa Vasorum by Plaque Type

Thin-Cap Fibroatheroma

Plaque Rupture

UlexStable Plaque

I

vWF

M

A

A

M

vWF

I

MSMCs

I

M

A

SMCs

SMCs

M

Ulex

I

M

I

A

A

I

ED

B C

H J

F

G

0

10

20

30

40

50

TCFA Rupture Stable

Th

NC

60P= 0.07 P= 0.01

NC

JACCIn Press

Adventitia

0

20

40

60

80

100

120

140

Normal PIT FA TCFA Rupture

Mea

n Ve

ssel

Den

sity

(# o

f ves

sels

/mm

2 )

Adventitia

0

20

40

60

80

100

120

140

Normal PIT FA TCFA Rupture

Mea

n Ve

ssel

Den

sity

(# o

f ves

sels

/mm

2 )

MAMA MA

FANormal PIT

MA

MA

Plaque RuptureTCFA

Adventitial Vasa Vasorum In Varying Plaque Morphologies(Ulex Europaeus)

Intimal - Medial Border

0

10

20

30

40

50

60

70

80

Normal PIT FA TCFA Rupture

Mea

n Ve

ssel

Den

sity

(# o

f ves

sels

/mm

2 )

Intimal - Medial Border

0

10

20

30

40

50

60

70

80

Normal PIT FA TCFA Rupture

Mea

n Ve

ssel

Den

sity

(# o

f ves

sels

/mm

2 )

FANormal PIT

M

M M

NC

MMTCFA

Intimal-Medial Border Vasa Vasorum in Varying Plaque Morphologies (CD31/CD34)

Plaque Rupture

Intimal-Medial Border T Cell Densities and % Macrophage Infiltration at Vaso Vasorum Hotspots in Varying Plaque Morpholgies

M

T Cell Densities (UCHL-1)

M

M

PIT Late FA Rupture

MM

Macrophage Infiltration (CD68)

M

T cellpericore

0

100

200

300

400

500

600

PIT Early Core Late Core TCFA Rupture

I-M border

0

100

200

300

400

500

600

700

800

900

normal PIT Early CoreLate Core TCFA Rupture

Neointima

0

100

200

300

400

500

600

700

800

normal PIT Early CoreLate Core TCFA Rupture

Adventitia

0

200

400

600

800

1000

1200

normal PIT Early Core Late Core TCFA Rupture

P=0.02

*

*P=0.0007 *

*

P<0.0001 P=0.20

* *

**

*=significant VS. normal

ConclusionsPlaques occur focally at branch points in the

presence of systemic risk factorsThe morphologic characteriestics most predictive

for the presence of unstable vs. stable plaque is necrotic core size, plaque area and to a lesser extent macrophage infiltration.

Intra plaque hemorrhages from leaky vv are responsible for enlargement of necrotic core, macrophage infiltration and progressive luminal narrowing

Non invasive detection of vulnerable plaques is the only mechanism through which morbidity and mortality for CAD can be reduced or eliminated.

Progression of stenosiscausingStable

Angina Pectoris

Asymptomaticprogression

ofstenosis

Acute Coronary Syndrome-Unstable Angina

-Myocardial Infarction-Sudden Coronary Death

Thrombosed Plaque

High Risk/Vulnerable Plaque

Asymptomatic Atherosclerosis

Normal Coronary Arteries

Over decades may develop

Which over years, may lead to

Which may progress, in an unpredictable manner to Which leads to

Development of atherosclerosis and Progression to ThrombosisDevelopment of atherosclerosis and Progression to Thrombosis

Severe stenosis

Occlusive Non-occlusiveNon-occlusiveErosion Plaque Rupture

Terminology for high risk and Vulnerable Coronary ArteryPlaque, Aug 29, 2003, Santorini, Greece

65%30% 5%

Chronic total occlusion

Erosion Plaque rupture

Calcified nodule

Normal Artery

Asymptomatic Atherosclerosis

Vulnerable plaque