Dr. Peter LIBBY

Pathophysiology of
Atherothrombosis
Inflammation in Atherosclerosis
Peter Libby
Brigham & Women!s s Hospital
Harvard Medical School
FAOD on Cardiovascular Biomarkers
Boston, MA October 1, 2007
Peter Libby, A Fire Within
Scientific American May 2002
Macrophage
Heterogeneity
in
Atherosclerosis
P. Libby Inflammation in atherosclerosis
Nature 2002;420:868-874.
Monocyte Heterogeneity in Mice
Ly-6C lo
Ly-6C hi
Ly-6C hi monocytosis is gradual and systemic
CD90/B220/DX5/
NK1.1/Ly-6G
Events
CD11b
Ly-6C
78± 7
Ly-6C lo
Chow
ApoE –/–
4 ±2 25±5
19± 6
Ly-6C hi
Western
Ly-6C lo Ly-6C hi Ly-6C lo Ly-6C hi
lo
22± 7 81± 6
Total
Monocytes
Ly-6C hi
Monocytes
Ly-6C lo
Monocytes
Cells/ml blood
Cells/ml blood
Cells/ml blood
2_10 6
10 6 0
2_10 6
10 6 0
2_10 6
10 6 0
Chow
DT (d): 63–86
DT (d): 63–86
DT (d): 59-94
ApoE –/–
Western
DT (d): 41–62
DT (d): 33–38
DT (d): 145-256
Diet (d):
0
300
0 300
Swirski, F. et al. J. Clin. Invest. 2007;117:195-205

Mast Cells in Atherosclerosis
Mast-cell deficiency mutes
mouse atherogeneisis
(26 weeks on an atherogenic diet)
Libby P. Inflammation in Atherosclerosis Nature 420:868 (2002)
Sun et al., Nature Medicine June 2007
Possible RolesR
of Mast Cells in Atherothrombosis
Inflammation
in Atherosclerosis
Peter Libby, A Fire Within
Scientific American May 2002
The traditional
“plumber!s s view” of
the complications of
atherosclerosis

MIs often arise from
non-critical stenoses
! Post-thrombolysis
angiography
! Serial angiographic studies
! Intravascular Ultrasound
studies
P Libby
What mechanisms may
cause acute coronary
syndromes in the
absence of critical
stenosis?
Four Mechanisms of Atherosclerotic Plaque Disruption
Libby & Théroux, Circulation, 2005
Fibrous
Cap
M.L. Higuchi
Lipid Core
Thrombus
Plaque
Rupture
Structural Integrity of the
Plaque!s s Fibrous Cap
• Depends on interstitial
collagen fibrils (types I
& III) synthesized by
smooth muscle cells
Interstitial Collagenases
MMP-1, MMP-8, & MMP-13
3/4 fragment
Gly 775 - Leu/Ile
776
1/4 fragment
Type I
Collagen
Cleaved
Type I
Collagen
Neoepitope

Cleaved Collagen Colocalizes with MMP-1 &
MMP-13 in Human Atheroma
lumen
lumen
cleaved type I collagen
lumen
MMP-1
type I collagen
lumen
MMP-13
GK Sukhova et al.
Circulation 99:2503 (1999)
Collagenase 3
Cell Type
(MMP-8)
Inflammation and the integrity
of the plaque!s fibrous cap
Lipid core
Synthesis
IL-1
TNF-"
MCP-1
M-CSF
IFN-#
–
CD-40L
+
+ + + +
Breakdown
Collagenase
Gelatinases
Stromelysin
Other proteases
+ pepidases
+
Fibrous
Cap
After Libby P. Circulation 1995
Collagenase-resistant mutant mice
•Mutation at the specific collagenase cleavage
site on type I collagen (“knock-in”)
collagenase cleavage site
GLY (775) - ILE (776)
GLY (775) - PRO (776)
(Zhao W, Byrne MH, Boyce BF, Krane SM. JCI 1999)
Collagenase-resistance promotes collagen
accumulation in mouse atheroma
Picro-sirius red staining with
polarization
Col +/+ / apoE -/- Col R/R / apoE -/-
Fukumoto, Deguchi, Libby, Rabkin, Sakata, Chin, Hill, Lawler, Varo, Schoen, Krane, Aikawa
Circulation 2004;110:1953
MMP-13/collagenase
collagenase-3 deficiency increases
fibrillar collagen in mouse atheromata
Mmp-13
+/+ /apoE
-/- Mmp-13
-/- /apoE
-/-
Bright-field illumination
Mmp-13
+/+ /apoE
-/- Mmp-13
-/- /apoE
-/-
Polarized light
Picrosirius red staining
Slide 33
Deguchi et al. Circulation 2005; 112:2708
(mm 2 )
Collagen area
0.2
p

Plaque rupture with thrombosis
Thrombus
Fibrous
cap
Thrombosis on a disrupted
atheroma, the cause of most acute
coronary syndromes, results from:
FJ Schoen, BWH
Lipid
core
• weakening of
the fibrous cap
thrombogenicity
of the lipid core
CD40 and Tissue Factor in Atheroma
Molecular Biology of the
High-Risk Plaque
Synthesis
Breakdown
IFN-#
–
Collagen-degrading
Proteinases
Fibrous
cap
CD40
Tissue Factor
Mach et al. Circulation. 1997;96:396.
Lipid core
CD-40L
+
After Libby P. Circulation 1995
+ + + +
+
IL-1
TNF-"
MCP-1
M-CSF
Tissue Factor
Procoagaulant
!Does the Biology
of Atherosclerosis
Suggest Useful
Biomarkers?
Inflammation in Atherosclerosis
Initiation
Libby, Ridker, Maseri
Circulation 2002;
105:1135-1143
Progression
Complication

A New View of
Vascular Diseases
!Inflammation: a putative
“unified theory” of
atherosclerosis
pathogenesis
Translation of the basic
science to the clinic:
!Can we use
inflammatory markers
to sharpen
cardiovascular risk
prediction?
Pro-Inflammatory Pathways
ICAM-1
Selectins, HSPs, etc.
Endothelium
and other cells
after Libby, Ridker.
Circulation 1999;100:1148–1150.
Pro-Inflammatory Risk Factors
Primary Pro-Inflammatory Cytokines
( e.g., IL-1, TNF-" )
IL-6
“Messenger” Cytokine
Circulation
CRP
SAA
Liver
Human
endothelial
cells express
the interleukin-
1$ gene
inducibly
Endotoxin and tumor
necrosis factor induce
interleukin-1 gene
expression in adult
human vascular
endothelial cells
Libby et al. Am J Path
124:179-186 (1986)
Human smooth
muscle cells
express the
Tumor
Necrosis
Factor " gene
inducibly
Warner SJC, Libby P.
Human vascular smooth
muscle cells: Target for and
source of tumor necrosis
factor. J Immunol
1989;142:100-109.
Pro-Inflammatory Pathways
ICAM-1
Selectins, HSPs, etc.
Endothelium
and other cells
after Libby, Ridker.
Circulation 1999;100:1148–1150.
Pro-Inflammatory Risk Factors
Primary Pro-Inflammatory Cytokines
( e.g., IL-1, TNF-" )
IL-6
“Messenger” Cytokine
Circulation
CRP
SAA
Liver

VCAM-1 expression in rabbit aorta
3 weeks on atherogenic diet
Pro-Inflammatory Pathways
Pro-Inflammatory Risk Factors
ICAM-1
Selectins, HSPs, etc.
Endothelium
and other cells
Primary Pro-Inflammatory Cytokines
( e.g., IL-1, TNF-" )
IL-6
“Messenger” Cytokine
Circulation
CRP
SAA
Liver
Li et al. Arteriosclerosis 13:197; 1993
after Libby, Ridker.
Circulation 1999;100:1148–1150.
Proliferating or
interleukin-1
activated human
vascular smooth
muscle cells
secrete copious
interleukin-6.
Loppnow H, Libby P.
J Clin Invest
1990;85:731-738.
CD40/ CD40 Ligand Dyad
• 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
CD40
S. Kinlay 2002
CD40L
(CD154)
Schonbeck U, Libby P. Circ Res 2001; 89:1092-1103
Functional CD 40
ligand is expressed
on vascular
endothelial cells,
smooth muscle
cells, and
macrophages in
human atheroma
Mach F, Schönbeck U,
Sukhova GK, Bourcier T,
Bonnefoy J-Y, Pober JS, Libby P.
PNAS 94:1931 (1997)
Interruption of CD40 signaling in
hypercholesterolemic mice:
• Inhibits the formation of
atherosclerotic lesions
• Prevents the evolution of
established atheroma
• Fosters features of the plaque
associated with stability

Platelets Release Soluble CD40 Ligand
CD40L
S. Kinlay 2002 S. Kinlay 2002
Inflammation Biology of the
High-Risk Plaque
Synthesis
Breakdown
Unstimulated
Platelets
Stimulated
Platelets
• Activated platelets are a
rich source of sCD40L
• Statins may lower sCD40L
levels
sCD40L
Andre P, et al. Circulation 2002; 106:896
Schonbeck U, et al. Circulation 2002; 106:2888
IFN-#
Lipid core
CD-40L
+
After Libby P. Circulation 1995
–
Matrix-Degrading
Proteinases
+ + + +
+
IL-1
TNF-"
MCP-1
M-CSF
Tissue Factor
Procoagulant
Fibrous
cap
Matrix Metalloproteinase-9 in
Human Atheroma
Galis et al. J Clin Invest 94:2493 (1994)
Matrix
metalloproteinase
activity
in extracts
of human
atheroma
J Clin Invest
94:2493 (1994)
Plasma concentrations and
genetic variation of matrix
metalloproteinase 9 and
prognosis of patients with
cardiovascular disease
Blankenberg, Rupprecht, , Poirier, Bickel,
Smieja, Hafner, , Meyer, Cambien, Tiret;
AtheroGene Investigators.
Circulation. 2003;107:1579-85.
1127 CAD Pts
Plasma concentrations and genetic variation of matrix metalloproteinase 9
and prognosis of patients with cardiovascular disease. Blankenberg et al.
AtheroGene Investigators. Circulation. 2003;107:1579-85.

Inflammation Biology of the
High-Risk Plaque
Synthesis
IFN-#
Lipid core
CD-40L
+
After Libby P. Circulation 1995
–
+ + + +
Breakdown
Collagen-degrading
Proteinases
+
IL-1
TNF-"
MCP-1
M-CSF
Fibrous
cap
Myeloperoxidase
Superoxide
Peroxynitrite
Myeloperoxidase
! An enzyme released by activated
granulocytes and a monocyte
subpopulation
! Bound to extracellular matrix at
sites of inflammation
! Converts Cl - + H 2 O 2 to HOCl
(hypochlorous acid), an oxidant and
chlorinating species
Macrophage myeloperoxidase regulation in
human atherosclerosis: implications for
acute coronary syndromes
Inflammation and Thrombosis
Activated platelets produce inflammatory modulators
Platelet-derived growth factor
Platelet factor 4
CD 154 (CD40L)
RANTES (PMN chemokine)
Thrombospondin
Transforming growth factor-$
Nitric oxide
Sugiyama et al. Am J Pathol 2001; 158:879-891
Libby & Simon. Circulation 2001
Croce &, Libby Curr Opin Hematol. 2007
Study Objectives
• To identify genes responsible for acute
thrombotic complications of CAD
(transcriptional profiling of STEMI vs.
CAD)
• To develop novel biomarkers for
cardiovascular disease and new
targets 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.

The platelet transcriptome
provides a snapshot in time
Platelets circulate
for ~ 7 days
• Since platelets lack
nuclear DNA, but
retain megakaryocyte-
derived mRNAs, , the
platelet transcriptome
provides a novel
window on gene
expression preceding
acute coronary events.
Normal
(no CAD)
Cardiac Catheterization Laboratory
Brigham and Women’s s Hospital
Stable
Angina
Unstable
Angina
STEMI
RNA isolated from platelet-rich plasma from
anticoagulated whole blood (up to 50-100
ng/50 mL), amplified, and profiled
Patients with ST-segment-elevation
myocardial infacrction (STEMI) have higher
plasma 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 study
(255 case-control pairs) in population of
apparently healthy post-menopausal
women to assess the risk of first
cardiovascular event (CV death, non-fatal
MI, stroke) associated with base-line
plasma levels of MRP-8/14.
Ridker, PM et. al. NEJM 2005
MRP-8/14
independently
predicts CV risk in
the Womens’ Heath
Study
MRP-8/14 improved
risk-prediction models
based on total
cholesterol:HDL or
CRP testing alone.
Healy et al. Circulation 2006;113:2278-2284
Pro-Inflammatory Pathways
ICAM-1
Selectins, HSPs, etc.
Endothelium
and other cells
after Libby, Ridker.
Circulation 1999;100:1148–1150.
Pro-Inflammatory Risk Factors
Primary Pro-Inflammatory Cytokines
( e.g., IL-1, TNF-" )
IL-6
“Messenger” Cytokine
Circulation
CRP
SAA
Liver

The dominant emerging
biomarker of cardiovascular risk
C-reactive protein: CRP