Plaque Progression and Instability:Critical Insights from PathologyCritical Insights from Pathology

Renu Virmani, MDRenu Virmani, MDCVPath Institute

Gaithersburg, MD, USAg, ,

Non-Progressive and Progressive Coronary PlaquesCoronary Plaques

non-progressive progressive

adaptive intimalhi k i

Intimalh

pathologicintimalthi k i f

thin-capfibroatheroma

NC

thickening xanthoma thickening fibroatheroma fibroatheroma

NC FC

lipid pool necrotic corep p

early late necrosis

Lesions with ThrombiLesions with Thrombi

• Plaque Rupture• Plaque Rupture• Plaque ErosionPlaque Erosion• Calcified NoduleCalcified Nodule

Causes of Coronary ThrombosisCauses of Coronary ThrombosisCalcified noduleCalcified noduleRuptureRupture ErosionErosion

RuptureSite

NCTh

ThThNC

Site

ThTh NCTh

Th Th

Th

Th

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

Gross and Light MicroscopicA

NC g pFeatures of Plaque RuptureTh

60-65% of Thrombi in Sudden Coronary Death occur

NC D FB

form Plaque Rupture

ThTh

NC D F

T cells SMCs

ThTh

T cells SMCs

C E G

NC

ThMΦ HLA‐DR

Fig 3-1

Th

Plaque erosion in a 33 year old female complaining of

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 CB

NC

ED F

MΦSMCs

T‐cells PLT FibrinT cells PLT Fibrin

Calcified NoduleBA

Calcified NoduleBA

*

**

**NoduleNodule*

*

**

*

Fibrin

CalcifiedCalcifiedplate

Frequency 2-7% of SCD, Older individuals, usually Men, Frequency 2 7% of SCD, Older individuals, usually Men, T2D and Prediabetes, equally common in tortuous right and left coronary arteries

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

Plaque rupture Plaque erosion Calcified noduleCalcified nodule

Th

LumenLumen

ThTh

Necrotic coreTh

60% thrombi in SCDM>F, Older, Ca++Eccentric = concentricG % i

30-35% thrombi in SCDM=F, youngerUsually eccentricL % i

2-7% thrombi in SCD, calcified platesM>F, older, mid RCA, LADU ll iGreater % stenosis

Macs, T cells, HLA-DR

Lesser % stenosisSMC rich, proteoglycans

Usually eccentricStenosis variableNodules of bone

Plaque rupture with mild stenosis and nonPlaque rupture with mild stenosis and non‐‐

Proximal

Plaque rupture with mild stenosis and nonPlaque rupture with mild stenosis and non‐‐occlusive thrombus: a mechanism by which occlusive thrombus: a mechanism by which plaques progress from an asymptomatic to plaques progress from an asymptomatic to  Th

symptomatic phasesymptomatic phase9mm

Rupture site 

Th

Th

3mm6mm

Th

NC

Healed Healed lesions lead to plaque lesions lead to plaque progressionprogressionHealed plaque rupture

NC NC

Multiple healed rupturep p

1

2

Mean % stenosis increases with number of prior rupture sites

is

80

90

90100

is

% s

teno

s

50

60

70

60

7080

% s

teno

s

Mea

n %

20

30

40

304050

Mea

n %

M

0

10

20

1 2 3 40

10

20

M

Number of prior ruptures, healed rupture sites

1 2 3 4A 0 1 2 3 4

Number of prior ruptures, acute rupture sites

B

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

IntimalIntimal IntimalIntimal FibrousPathologicintimal Thin cap

Progression of Human Coronary AtherosclerosisVirmani R, et al. ATVB2000;20:1262

IntimalIntimalthickeningthickening

IntimalIntimalxanthomaxanthoma

Fibrouscap atheroma

intimal thickening

Thin‐capFibroatheroma

NC

NCEL FC

Calcified noduleRuptureErosion

NoduleNCHealed Rupture

ThThThTh NCNCTh

Nodule

FCTh

C 2+

ThTh

Ca2+

ThTh ThThThTh

Development of Necrotic Corep

The Necrotic Core“graveyard of dead M s”graveyard of dead M s

Thr

NC

Ruptured plaque atRuptured plaque at area of thinned fibrous cap

inflammation

Necrotic Core

stress on fibrous coppCoagulation thrombosis

proteases

T

Adaptive Intimal Thickening

P th l i I ti l Thi k iSmooth muscle cell

proliferationdeath (apoptosis MacrophagesMacrophages

Pathologic Intimal Thickening

death (apoptosismicrocalcification

Extracellular lipid (lipid pool) ± luminal macrophages

MacrophagesMacrophages

Inflammation – T-cells Macrophage

Infiltration into LP,apoptosis

Fibroatheroma ( ± calcification)

(early and late)Macrophage infiltration

“Fatty streak”“Fatty streak”Thin cap fibroatheroma

Macrophage infiltration(proteolytic enzymes)

Hemorrhage (red cell membrane)

Plaque rupture

Associated with lesion regression

Microcalcificationof macrophages + iron Flow disturbances

Lesion enlargement – asymptomatic or symptomatic

Histomorphometric Analysis of Progressive Coronary LesionsCoronary Lesions

Pit (no Macs)) Pit (+ Macs)) Early FA) Late FA)

Differential Expression of Differential Expression of HyaluronanHyaluronan andandVersicanVersican in the Developing Necrotic Corein the Developing Necrotic Core

Fibroatheroma ‘Early’ Necrosis

VersicanVersican in the Developing Necrotic Corein the Developing Necrotic CoreA B

Fibroatheroma  Early  Necrosis

NC

MΦ bHABR Versican

C D

Thin‐cap Fibroatheroma ‘Late’ Necrosis

NC

bHABR VersicanMΦ

Histomorphometric Analysis of Plaque Componentp

Pit (no Macs)) Pit (+ Macs)) Early FA) Late FA)

Macrophage (KP-1.CD68))

In-Situ End Labeling (DNA fragmentation, apoptosis))

Phagocytosis efficiency of apoptotic cells (AC) in advanced atherosclerotic plaque and human tonsils

Carotid plaque Tonsil

TUNNEL (AC, red)

CD 68CD 68 (macrophages, blue)

TonsilAtherosclerotic plaque

100 The radio of free ACversus phagocytized AC was 19 timeshi h i th

100

80

60

100

80

60

tic cells

phages*

***

higher in athero‐sclerotic plaques as compared to human tonsil

40

20

40

20

0

% apo

ptot

% m

acrop

******

***

*

human tonsil

Free        uptake                      1AC      2AC     >2AC

0 0

Schrijvers DM et al. ATVB,2007

Morphologic Characteristics of Plaque Rupture and Thin‐cap Fibroatheromas

Plaque typeNecroticCore (%)

Fibrous capThickness (µm)

MΦs(%)

SMCs(%)

T‐lymph

CalcificationScorePlaque type Core (%) Thickness (µm) (%) (%) lymph Score

Rupture 34±17 23±19 26±20 0.002±0.004 4.9±4.3 1.53±1.03

Thin‐capFibroatheroma 23±17 <65µm 14±10 6.6±10.4 6.6±10.4 0.97±1.1

P value 0.01 0.005

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

ns ns 0.014

SMCs  smooth muscle cells, T lymph  T lymphocytes

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

Remodeling in Varying CoronaryLesion MorphologiesLesion Morphologies

A.

5

4

NCMedial SMC loss

pected

 IEL

ue area)

4

3

2

1

IEL‐Exp

(/plaq 0

‐1

‐2

osion

Stab

le

oma

hage

pture

pture

sion

‐3

Ero S

n cap athe

ro

que he

morrh

Acute rup

Healed rup

Total occlus

Medial SMCapoptosis

Thi

Pla q

Thi Fib th A Pl R

Frequency and Location of Unstable Lesions: Thin-cap Atheromas, Acute and Healed Ruptures in the Coronary Circulation

504540

Thin-cap Fibroatheromaer

oma’

%)

Acute Plaque Rupture

’

504540

3156

40353025Fi

broa

thqu

ency

(%

Rup

ture

’en

cy (%

) 40353025 17 1526

Total Lesions= 68Total Lesions= 131

2015105Th

in-C

ap

Freq

‘Pla

que

Freq

ue 2015105

1526 23

11

5 650

‘T

Healed Plaque Rupture

50

50 pLAD

1 2 1 15 62 2

61

ture

’(%

)

50454035

pLADpRCpLCxLM

Total Lesions= 136

61

aled

Rup

teq

uenc

y 30252015

LOMdRCdLADdLCx

Total Lesions= 136

23 23

‘Hea Fr

151050

126 6 4 1 Kolodgie et al. Current Opinion

in Cardiology 2001;16:285

Intraplaque HemorrhageIntraplaque Hemorrhage, Oxidant Stress & Plaque Vulnerability

A Evidence that Human Coronary Plaques Express a Latent Proangiogenic Phenotype

I

MNormal artery with adventitial Vv Fibroatheroma with severe

Intraplaque hemorrhageULEX E.

B

MIFibroatheroma with

HemNCMITortuous and Abnormal Vv

HemNC

C

Fibroatheroma withLeaky Vv ( i l h h )(peri‐vascular hemorrhage)

)

Thin‐cap FibroatheromaRecent Intraplaque Hemorrhage is seen atRecent Intraplaque Hemorrhage is seen at 

Multiple sites in Patients Dying SCDFrequency of Plaque

A BFrequency of Plaque 

Hemorrhage

a bbrupture erosion Severe CAD

(>75%)

Morphometric Analysis of Hemorrhagic Events in Human

Hemorrhagic Pericarditis

GpA MΦVulnerable Plaque

WFFMΦNC

G A

Plaque TypeGpAScore Iron

Necrotic Core(mm2)

MΦ(mm2)

vWFFeMΦ GpA

PIT no core(n=129)FA early core(n 79)

( ) ( )0.09±0.04

0.23±0.07

0.07±0.05

0.17±0.08

0.0

0.06±0.02

0.002±0.001

0.018±0.004(n=79)FA late core(n=105)TCFA(n=52)

*0.94±0.11

*1 60±0 20

*0.41±0.09

*1 24±0 24

*0.84±0.08

*1 95±0 30

*0.059±0.007

*0 142±0 016(n=52) 1.60±0.20 1.24±0.24

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

1.95±0.30 0.142±0.016

Kolodgie FD, et al. New Engl J Med 2003

Necrotic Core FormationMechanism(s) and MoleculesMechanism(s) and Molecules

Early Necrosis

• Early foam cell apoptosis (via ER stress path

engulfment:y p p ( p

• Clearance by phagocytosis (efferocytosis)

d f ti lf t ( )defective engulfment: (molecular genetic causation studies in mice)

Late Necrosis Hemorrhagic NecrosisExcess foam cell apoptosis

Defective efferocytosis

Excess• free cholesterol• free hemoglobin (Hb)y

1) Fas ligand (apoptosis stimulating fragment)

2) transglutaminase-2 3) lactadherin

g ( )• macrophages

efferocytosis3) lactadherin4) Mertk (Mer receptor tyrosine kinase)

efferocytosis

Role of Vasa Vasorum in the leakage of RBC into the plaque d h ti tiand macrophage activation

vWf ‐‐VEGF + ang‐1 vWf

iNOS0202

HboxLDL

Lp‐PLA2

HbiNOS

HO‐1

FeHaptoglobin

ICAM

A t ti

oxLDLCD163

CeroidHaptoglobin1‐1 or 1‐2 genotype

Apoptotic bodies

oxRBC↑IL‐6+MCP‐1

Free CholesteroloxLDL

↓phagocytosis Haptoglobin

↓phagocytosis of apoptotic bodies

Foam cellsoxLDLoxRBC

p g yGeneotype 2‐2 vs.1‐1 or 1‐2associated with greater iron in plaque (46% vs.12%) among T2D

Development of Calcified Nodulep

Patients dying  from Sudden coronary Death:Extent of Coronary calcification by decades

80

100

120um

sco

re

BlacksWhites

2.5

3

nsc

ore

40

60

80

ary

calc

iu

1.5

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calc

ifica

tion

0

20

40

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on

0

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0

thirt

ies

fort

ies

fiftie

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sixt

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oath

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a

Thin

cap

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gic

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fibro

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Path

olo

thic

ken

Radiograph Movat Stain Ground Section (40 µm) 

Ca++Ca++

Eruptive Calcified Nodule with Thrombosis in a Heavily 

LMy

Calcified Coronary Artery

LAD LCX

RCA

LAD LCX

66 ld hit66 year old white manH/O hypertension, prediabetic and BMI 34 7LM prox LCX

prediabetic, and BMI 34.7 LM

Th

Eruptive Calcified Nodule with ThrombosisA B CCalcified plate

Fibrous cap

Nodule

CD61Movat 100 μm1.0 mmMovat 200 μm

Th

D E F

Osteoclast-like cell

N d l

CD68Fibrin II H&E 200 μm100 μm100 μm

Nodule Nodule

The Role of  Risk Factors in SCD patients St bl C l ifi d

ControlN=163

Stableplaquen=131

Rupturen=84

Erosionn=45

Calcified nodulesN=8

Age 46 54 49 45 57

Male 118 (72%) 102 (78%) 78 (90%) 29 (64%) 6 (88%)

R %Bl k 75 (46%) 46 (35%) 21 (24%) 16 (36%) 2 (25%)Race %Black 75 (46%) 46 (35%) 21 (24%) 16 (36%) 2 (25%)

BMI 28.4 28.3 29.4 26.7 31.8

HbA1c 6 6 7 5 7 2 7 0 7 0*HbA1c 6.6 7.5 7.2 7.0 7.0*

Hx of HTN 35 (21%) 61 (47%) 27 (31%) 9 (20%) 5 (63%)

Smoker 64 (39%) 63 (48%) 53 (60%) 33 (73%) 7 (88%)( ) ( ) ( ) ( ) ( )

T. Chol 202 208 258 211 208

HDL 46 41 37 39 42

T.Chol/HDL 5.3 5.8 7.7 5.9 5.4

Healed MI 1 (1%) 76 (58%) 37 (42%) 11 (24%) 4 (50%)

Plaque Burden ‐‐‐ 232.4 248.1 178.9 258

* Out of 8  case, 4 were DM, and 2 were Pre‐DM.

Distribution of Atherosclerotic Coronary Disease

LAD                                              LCx RCA

Calcified nodule with thrombusNodular calcificationFibocalcific plaqueThin‐cap fibroatheromaTotal occlusionTotal occlusionPathological intimal thickening

A C

Nodular Calcification without ThrombosisA C

B D

Plaque RuptureHealed Plaque 

RuptureFibrocalcific

PlaquePlaque Fissure

Th

C l ifi d

Calcified Nodule with l i l th b

Calcified Plate F t ti

Nodular C l ifi ti

Calcifiedplate

luminal thrombusFragmentation Calcification

Fibrin

Calcified Plate Nodules of calcium Medial 

Fibrin

Luminal thrombusfragmenationfrom artery torturocityand beating heart 

Nodules of calciumDestroying the media destruction

Luminal thrombussurrounded by Fibrin and platelet

Inflammation: The Road toPl P iPlaque Progression

Summary:• The earliest lesion of plaque progression is Pathologic Intimal

thickening

Summary:

thickening.• PIT, lipid pool is converted to necrotic core from

macrophages infiltration and apoptosis leading to earlymacrophages infiltration and apoptosis leading to early necrotic core formation.

• Late necrotic core is likely the result of defective efferocytosisy yas well as plaque hemorrhage which contribute to free cholesterol within necrotic core

• Inflammation continues to increase as plaques progress and is maximum in plaque rupture.

Summary: Thrombosis( )Plaque rupture is a main cause of thrombosis (65‐70%), 

while other minor causes include erosion (30%) and calcified nodule (4 7%)calcified nodule (4‐7%).

Vulnerable plaques (TCFA) is a likely precursor lesions of rupture Macrophage infiltration play an important role inrupture. Macrophage infiltration play an important role in modification of plaque vulnerability.

Plaque hemorrhage from “leaky” vasa vasorum is anPlaque hemorrhage from  leaky  vasa vasorum is an important contributor to the enlargement of the necrotic corecore.

Calcified nodules  is another substrate for thrombosis, especially in elderly male individuals with high plaqueespecially in elderly male individuals with high plaque burden, tortuous arteries, diabetes , metabolic syndrome, hypertension, and smoking.yp , g

A k l d t• Masataka Nakano, M.D.

Acknowledgments• Fumiyuki Otsuka, M.D.

• Frank Kolodgie, Ph.D.

• Aloke V. Finn, M.D.

• Elena Ladich, M.D.

• Ed Acampado, D.V.M.

• Robert Kutz, M.S.,

• You‐hui Liang, M.D.

• Erica Pacheco, M.S.,

• Hedwig Avallone

• Lila AdamsLila Adams

• Russ Jones

• Abebe AtisoAbebe Atiso

• Rosalind Mathew