thrombosis and inflammation, next target for new drugs...
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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