Thrombosis and Inflammation in Plaque Vulnerability:in Plaque Vulnerability:

What is the Next Target for New Drugs?

Dr. Renu Virmani, MDDr. Renu Virmani, MDCVPath Institute

G ith b MD USAGaithersburg, MD, USA

Natural History of Atherosclerosis• Systemic factors –

Dyslipidemia, diabetes mellitus, smoking, hypertension, age and gender, hsCRP, Lp‐PLA2, etc.

• Local factors –Branch points, e.g. carotid bifurcation, abdominal aorta just 

above bifurcation, coronary branch point, and arch vessels at take off are the sites of early atherosclerosistake off, are the sites of early atherosclerosis .

• Thrombosis –Occurs in the coronary arteries at focal points and is mostOccurs 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), y ( y g p y ),and occur at sites where there are underlying plaque characteristic that result in thrombosis.

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

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

The Role of  Risk Factors in SCD patients 

Sudden Coronary DeathN=264

ControlN=163

Stable plaquen=131

Rupturen=88

Erosionn=45

P value(ANOVA)

Age 45.6 54.0* 48.8 45.0 <0.0001

Male 118 (72%) 102 (78%) 78 (90%) 29 (64%) 0.0032

Race %Black 75 (46%) 46 (35%) 21 (24%) 16 (36%) 0.0060

BMI 28 4 28 3 29 4 26 7 0 21BMI 28.4 28.3 29.4 26.7 0.21

Hx of DM 8 (5%)  28 (21%) 6 (7%) 4 (9%) <0.0001

I li 5 (3%) 21 (16%) 6 (7%) 1 (2%) 0 0002Insulin use 5 (3%) 21 (16%) 6 (7%) 1 (2%) 0.0002

HbA1c 6.6 7.5* 7.2 7.0 0.0043

* Significantly different from Control by post‐hoc analysis

Extent of Coronary Artery Disease in SCD patients 

ControlN=163

Stable plaqueN=131

RuptureN=88

ErosionN=45

P value(ANOVA)

Hx of HTN 35 (21%) 61 (47%) 27 (31%) 9 (20%) <0.0001

Smoker 64 (39%) 63 (48%) 53 (60%) 33 (73%) <0 0001Smoker 64 (39%) 63 (48%) 53 (60%) 33 (73%) <0.0001

T. Chol 202.2 208.7 257.8* 210.8 <0.0001

HDL 45 9 41 4 36 5* 39 0 0 0018HDL 45.9 41.4 36.5* 39.0 0.0018

T.Chol/HDL 5.3 5.8 7.7* 5.9 <0.0001

TC/HDL 5 69 (42%) 70 (53%) 77 (88%) 24 (53%) 0 0001TC/HDL>5 69 (42%) 70 (53%) 77 (88%) 24 (53%) <0.0001

Heart Wt 457.4 493.9* 495.1 428.0 0.0014

Healed MI 1 (1%) 76 (58%) 37 (42%) 11 (24%) <0.0001

Plaque Burden ‐‐‐ 232.4 248.1 178.9 <0.0001

* Significantly different from Control by post‐hoc analysis

Attributes Odds CI P value

Independent risk factors for SD from Stable Plaque 

HbA1c 1.26 1.10‐1.45 0.0007

T. Chol/HDL 1.03 0.93‐1.14 0.58

Hypertension 2 65 1 47‐4 76 0 0012Hypertension 2.65 1.47 4.76 0.0012

Smoker 1.85 1.08‐3.18 0.026

Independent risk factors for SD from Plaque Ruptureder, race

Attributes Odds CI P value

HbA1c 1.16 1.01‐1.34 0.041

Independent risk factors for SD from Plaque Rupture 

age, gen

d

T. Chol/HDL 1.29 1.15‐1.45 <0.0001

Hypertension 1.69 0.83‐3.44 0.15

S k 2 74 2 46 5 13 0 0017justed

 by 

Smoker 2.74 2.46‐5.13 0.0017

Attributes Odds CI P value

Independent risk factors for SD from Plaque erosion 

Adj

Attributes Odds CI P value

HbA1c 1.13 0.95‐1.36 0.17

T. Chol/HDL 1.07 0.94‐1.22 0.29

7 of 66

Hypertension 1.34 0.47‐2.78 0.78

Smoker 4.93 2.29‐10.64 <0.0001

Extent of Coronary Artery Disease 

in SCD patients with and without DM • More likely to have CAD• Increased severityp • Increased severity

P=0.04(%) (%)

P=0.005

ents

n=63

n=206

n=137 n=16n=19

n=28 n=88

n=54n=64

e of

Pat

ie

n=19

n 137 n 16

P 0 04 P 0 0001erce

ntag

e

(%)(%)

P=0.04 P=0.0001Pe

n 24

n=108n=98

n=39

19

n=36

n=120n=24

CTO20

42

56n=14

n=86

(Type II)

Diabetes and Pre-Diabetes Are Present In Most MI patientsConsecutive patients presenting with an AMI, n = 181

20%

Oral Glucose Tolerance Test25%25%

20% 40%40%35%35%

80%DiabeticDiabeticNo DM by Hx DM

Prediabetes

Norhammar A, et al., Lancet. 2002;359:2140‐44.Normal

Undiagnosed Prediabetes and T2DUndiagnosed Prediabetes and T2D

One‐third of patients do not survive their first MI. 

Exponential increase in obesity, pre‐diabetes, T2D. 

I i id ifi i f di d T2D i CVDIncreasing identification of undiagnosed T2D in CVD. 

What is the prevalence of undiagnosed T2D and pre‐diabetes in individuals having p gsudden death?

Schneiderman, Virmani, Plutzky et al, in review

Incidence of Undiagnosed T2D in S dd C D th

~1/3 patients die from first heart attackSudden Coronary Death

pIncidence of undiagnosed T2D in sudden death?ADA criteria for dx of T2D (>6.5 HbA1c), pre‐DM (>5.7 to <6.5)

32%32%580 cases

38%38%30%30%

580 cases Out of hospital deathsAutopsy seriesA1CA1C

T2D (based on A1C >6.5) Prediabetes

Schneiderman, Virmani, Plutzky et alNormal Prediabetes

Development of Necrotic Core

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

ApoB‐lipoprotein (ApoB‐LP) Promotes Monocyte Recruitment and Subsequent Foam Cell Formation  

Proteoglycans

Moore, Tabas . Cell. 2011;145:341‐355sPLA2 = secretory phospholipase A2S‐SMase = secretory sphingomylinase

A B

Plaque ProgressionPlaque Progression

Pathologic Intima Thickening Fibroatheroma ‘Early’ Core

A. B.

NCLP

CD68

NCLP

CD68

LP

Fibroatheroma ‘Late’ Core Thin Cap Fibroatheroma

C. D.

p

CD68

NCNC

CD68 CD68CD68

Mechanism of Necrotic Core ProgressionEarly Late HemorrhagicA. B. C.y g

FreeNC NC NC

Free-CholCD68 CD68

HP-2

ICAMApoptotic Body (AB)

HP-2

CD163Engulfment Defectiveengulfment

Macrophage-engulfed

Free ABs(red)

Hemorrhagein NC

GpA

p g gABs (red)

Independent risk factors for “rupture”Independent Morphological Predictor of Rupture vs. TCFA

ovat

Mo

e)CD

68 

acropahge

(Ma

P Value Odds Ratio* 95% CI

NC Area 0.02 2.73 1.14 – 6.30

C thi k 0 02 0 44 0 23 0 82Cap thickness 0.02 0.44 0.23 – 0.82

Macrophage Area 0.07 1.71 0.96 – 3.03*adjusted by S.D.

Proatherogenic Properties of Lipoprotein-Associated Phospholipase A (Lp PLA )Associated Phospholipase A2 (Lp-PLA2)

Lp PLA C kiLDL

Lp‐PLA2

ROSMMPs

Cytokines

apoB

ActivatedEndothelial cells

M t dh

+HO‐1

apoB

Extracellular matrix

§ § § § §Monocyte adherance

Subendothelial

Lp‐PLA2

Oxidized LDL

+

Macrophage/foam cellsCD36

L PC NEFA

Lp‐PLA2+

Necrotic core LDL oxidation

02‐

Lyso‐PC + oxNEFA

+

ExpansionoxPC

oxPCoxPS

Apoptosis/Necrosis

Lysophosphatidylcholine (Lyso‐PC)Oxidized nonesterified fatty acid (oxNEFA)Heme oxygenase (HO‐1)

Wilensky RL, Macphee CH Curr Opin Lipidol2005;16:442‐446 & 2009,20:415‐420

Caslake MJ, Packard CJ. Nat clin pract2005;2:529

oxPSoxPC

Pathologic Intimal thickening FibroatheromaA B

Lp PLA Lp PLALP

NC

Lp‐PLA2 Lp‐PLA2

Thin Cap Fibroatheroma Plaque RuptureDC

ThNC

NC

Lp‐PLA2 Lp‐PLA2

35E.

% Lp-PLA2 Staining in

30

20

25

**

% Lp PLA2 Staining in Varying Coronary Plaque

Morphologies 15

20

10P= ns *

Lp‐PLA2 (‐)

Lp-PLA2 is strongly expressed within the necrotic core and

PIT FA TCFA Rupture

5

0 Kolodgie et al, ATVB, 2006;26:2523-2529

surrounding macrophages of vulnerable and ruptured plaques.

Histomorphometric Analysis Stratified by Lesion Type1010

8

10Macrophages (%) Apoptotic Cells /mm2 Lp-PLA2 Score

p=0.0023 p=0.0004 p=0.0035

3

8

10

4

62

4

6

2

41

2

4

0

Fatty Streak PIT without Mac PIT with Mac Early Core Late Core

00

Lesion Type (n=53) Plaque Area (mm2)

Stenosis(%)

NC Area (mm2)

Macrophages (%)

Apoptotic Cells /mm2

Lp-PLA2 Score

Fatty Streak (n=6) 1.45±0.78 41.3±22.2 1.09±0.48 1.71±1.91 1.00±0.84

PIT without Mac (n=8) 3.86±2.12 41.9±12.3 0.71±0.80 2.07±1.16 0.25±0.27

PIT with Mac (n=15) 2.42±0.96 45.2±14.6 1.51±1.29 2.35±1.72 1.27±0.90

Early Fibroatheroma (n=14) 4.39±1.79 60.3±12.4 1.03±1.03 1.50±0.85 3.76±2.00 1.29±0.58

Late Fibroatheroma (n=10) 4.77±1.69 69.7±14.5 1.24±0.67 4.39±4.10 5.82±2.92 1.55±0.60P value 0.0002 0.0002 0.5961 0.0023 0.0004 0.0035

Co-Expression of LP-PLA2 with Apoptotic Macrophages in Early Necrotic Core

LP-PLA2 Immunostaining (Bluish-Grey) plus TUNEL (Red)

Angiogenesis and inflammation in the progressive enlargement of the necrotic core p g g

Adventitia & Intraplaque Vasa VasorumAdventitia & Intraplaque Vasa Vasorum150 μm thick sections stained with Ulex

Finn AV, et al. Arteriosclero Thrombosis ,Vascular Biology 2008

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)

Modified from Jain et al., Nat Clin Pract Cardiovasc Med, 2007)

The Relationship of Plaque Thickness and Vasa VasorumDensity in Progressive Human Coronary Lesions

Quartile 1 (n=9) <0.50 mm

Quartile 2 (n=15) 0.50 mm ‐ 0.99 mm

Quartile 3 (n=26) 1 00 mm 1 50 mmQuartile 3 (n=26) 1.00 mm ‐ 1.50 mm

Quartile 4 (n=23) >1.50 mmP = <0.0001

R2 = 0.9721

Quartile 1 (n=9)

FA (early core)AIT PIT FA (late core) TCFA Plaque Rupture

Quartile 2 (n=11) Quartile 3 (n=25) Quartile 4 (n=19)

Vaso Vasorum Density and Inflammatory Cells

80

100

800

1000

T cell Vaso Vasorum

80

100

200

250

B cell Vaso Vasorum(/mm2) (/mm2) (/mm2)

40

60

400

600

40

60

100

150

0

20

0

200

Normal PIT Early FA Late FA TCFA Rupture

0

20

0

50

Normal PIT Early FA Late FA TCFA Rupture

Mechanisms of coordinated angiogenesis and inflammation in the progressive enlargement of the necrotic core 

T‐lymphocyte accumulation in the 

I Vv

media causing destruction of medial wall along with  induction of angiogenesis

Vv

Vvangiogenesis

TT

MVv

IEL

T

TT T

T

AVv

VvVv

EEL

BB

B B

BB B

BB

TVv

↑↑B B T‐lymphocytes ‐ ↑angiogenesis via

Toll‐like receptors (TLR) 2 and 4,CD40/CD40L 

↑Macrophage via MCP=1, M‐CSF within plaque ‐↑VEGF =↑angiogenesis

Macrophage Diversity around Angiogenesis, Plaque Hemorrhage 

and Iron depositsand Iron deposits 

Signaling Pathways in Macrophage g g y p gAtherosclerosis

LipoproteinsLipoproteins

Hydolysed

Moore et al. Cell. 2011;145:341‐355

Expression of Haptoglobin Receptor (CD163) on a Subset of Macrophages in Human Coronary Plaque Rupture

Modified from: Boyle et al., AJP 2009;174:1097

ges

(%)

Mac

roph

a

HLADRCD163

+-

-+

++

--

MNC NC

Th

NCNC

CD163_FeCD68 CD68

CD68 CD36 ABCA1CD163

M(Hb)

Different Phenotype of Macrophage SubclassORO

M(Hb)

Foamy

M1(F )(Foamy) 

NCM(Hb) 

NC

*

*este

rol

hole

ster

ol)

20

-10

0

10

-stim

ulat

ed c

hole

(% o

f cel

lula

r ch

-50

-40

-30

-20TotalFreeEsterified

Apo

AI-

redu

ctio

n -60

50

Control Hb:Hp

Classically activated macrophage+ Pro‐inflammatory cytokines production+ Antigen presentation & microbicidal activity

Macrophage DiversityM1 Macrophage

IFN‐Ƴ LPS

+ Antigen presentation & microbicidal activity+ Expression of MHC class II molecules

Pro‐inflammatory cytokines

MHC class II

MannoseƳ,IL‐6TNFIL‐1

y y Mannosereceptor

IL‐10FeROS

Hb:HpCD163FPN

Fe2+ ABCA1/ABCG1

Free

LXRM (Hb)

Monocyte M2 Macrophage

Mannose Receptor Upreglation

MHC class II

Free cholesterol/sterols

IL‐4, IL‐13

Mannose Receptor Upreglation

IL‐10IL‐1Ra

Anti‐inflammatory cytokinesHDL

Activated PPARƳAlternatively activated macrophage+ Anti‐inflammatory cytokine production+ Cell growth and tissue repairE d i i i+ Endocytic activity

Possible Next Targets for New Drugs M1 Macrophage

Pro‐inflammatory cytokines

MHC class II Statins, CETP inhibitorsl d bIL‐6

TNFIL‐1

y y

Darapladib – LpPLA2inhibitor Antiinflammatory drugs:

Mannose Receptor Upreglation

M2 Macrophage

Antiinflammatory drugs: Methotrexate, low dose; and IL1β antibody.

IL‐10IL‐1Ra

Anti‐inflammatory cytokines

β y

LDN 193189 (LDN) inhibits BMP signaling to prevent activation of

Mannosereceptor

IL‐10F 2+

Fe2

ROS

g g phepcidin transcription factors, leading to reduced hepcidin and maintenance of FPN in macrophages thereby

CD163FPNFe2+ ABCA1/AB

CG1Free cholesterol/sterols

LXRof FPN in macrophages, thereby reducing intracellular iron and ROS and increasing the expression of ABCA1 and ABCG1 to promote lipidols

HDLFPN= Ferroportin

ABCA1 and ABCG1 to promote lipid efflux and reduce foam cell formation.

Factors and Conditions Associated with Increased Risk for Acute Coronary Events (Circulation 2012)Risk for Acute Coronary Events (Circulation 2012)

Coronary Plaque Characteristics

Coronary Blood Flow Dynamics

Intrinsic Hemostasis Factors

Metabliic and Inflammatory Conditions

NeuroharmonalImbalance

EnviornmentalFactors and  DrugsCharacteristics Dynamics Factors Conditions Drugs

Plaque Burden Blood viscosity Platelet function/volume

DiabetesMellitus

Stress Somking

e

Lumen encroachmen

Shear stress Circadian variation

Obesity Catecholamine surges

Pollution

L i l ti R d d bl d F t V L id D li id i D i Cli tLesion location Reduced blood flow/ low CO

Factor V Leiden def

Dyslipidemia Depression Climate

Plaque composition

Vascular tone and reactivity

vonWilliebrandFactor def

Connective tissue

Exertion Legal drugscomposition and reactivity Factor def tissue 

diseases

Plaque biology Arterial hypertension

Antiphospho‐lipid syndrome

Infections Autonomic dysfunction

Illegal drugshypertension lipid syndrome dysfunction

Plaque configuration and remodeling

Renal Disease Endocrine imbalance

Diet

and remodeling

Endothelial dysfunction

Sedentary life

Summary: Thrombosis and InflammationPlaque rupture is a main cause of thrombosis (65‐70%), while other minorPlaque rupture is a main cause of thrombosis (65 70%), while other minor 

causes include erosion (30%) and calcified nodule (2‐5%). (Rivaroxaban a Xainhibitor was effective in reducing cummulative CV event rate in patients presenting with recent ACS (STEMI NSTEMI or UA)presenting with recent ACS (STEMI, NSTEMI, or UA) 

Risk factors are predictive of specific plaque types.

Diabetes and metabolic syndrome play an important role in CAD. (Better control)

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

E t t f h i filt ti d ti i di t f lExtent of macrophages infiltration and necrotic core size are predictors of plaque vulnerability. (Antiinflammatory drugs: Methotrexate, low dose; and IL1βantibody).

Lp‐PLA2 may be involved in plaque progression and necrotic core expansion, therefore the inhibition of this enzyme is a potential therapeutic approach to prevent plaque progression and stabilize atheromatous plaques. (Darapladib) p e e p aque p og ess o a d s ab e a e o a ous p aques ( a ap ad b)

Intraplaque hemorrhage from “leaky” vasa vasorum is an important contributor to necrotic core expansion and potential lesion instability. 

Macrophage subtypes may help us better understand the role of plaque hemorrhage and plaque stabilization vs. plaque rupture (LDN supresses hepcidinincreases macrophage cholesterol efflux and reduces foam cells).

AcknowledgmentsFunding

CVPath Instit te

FundingCVPath Institute Inc.

CVPath Institute, Inc.

CVPath InstituteMasataka Nakano, MDFumiyuki Otsuka, MD, PhD ,Saami Yazdani, PhDFrank D Kolodgie, PhDElena Ladich, MDS i Y d i PhDSaami Yazdani, PhDRuss JonesRobert Kutz, MSEd Acampado DVMEd Acampado, DVMYouhui Liang, MDAbebe Atiso, HTMichael Cooper

Washington DC

Michael CooperJinky BeyerGiselle MagsalinHedwig Avallone HTHedwig Avallone, HTLila Adams, HTHengying Ouyang, MD