This document discusses research on the roles of collagen and collagenases in atherosclerotic plaque vulnerability. It presents several hypotheses: 1) that human and experimental plaques contain collagenases, 2) that lipid lowering improves features associated with vulnerability by reducing macrophages and collagenase expression while increasing collagen, and 3) that collagenolysis regulates collagen accumulation in plaques of genetically altered mice. Several studies are described that provide evidence supporting these hypotheses, finding that collagenases are expressed in human and animal plaques, lipid lowering reduces vulnerability features in rabbits by altering collagenase and collagen levels, and collagenase-resistant mice have altered collagen in plaques. The research suggests collagenases critically regulate collagen metabolism in plaques and their clinical complications.

1. Masanori Aikawa, MD, PhD
Peter Libby, MD
Cardiovascular Medicine
Brigham and Women’s Hospital
Harvard Medical School
Boston, MA
The balance of power
in “vulnerable” plaques:
collagenases vs. collagen

2. Abstract
Rupture of the “vulnerable” plaque often triggers acute coronary
syndromes.
Interstitial collagen determines plaque stability.
We have demonstrated macrophage expression of collagenases
(MMP-1, MMP-8 and MMP-13) in human and experimental
atherosclerosis.
Collagen degradation by collagenases may contribute to the
vulnerability of atheroma.
Lipid lowering in hypercholesterolemic rabbits reduces collagenase
expression and ,in parallel, increases collagen in atheroma, suggesting
a critical role of collagenase in collagen balance.
We have recently found that collagenase-resistance alters collagen
content in mouse atheroma.
These results suggest that collagenase action regulates the collagen
metabolism crucial to the clinical complications of atheroma.

3. M.L. Higuchi
ThrombusThrombus
ThinThin
FibrousFibrous
CapCap
Lipid CoreLipid Core
Rupture of the “vulnerable” atheroscleroticRupture of the “vulnerable” atherosclerotic
plaque triggers acute coronary syndromesplaque triggers acute coronary syndromes

4. Atheromatous core
Lipid, Macrophage-rich
Proteolytic activity (MMPs)
Prothrombotic activity (TF)
Characterization of the “vulnerable”
atherosclerotic plaque
Lumen
More stable plaque
Adopted from Masanori Aikawa and Peter Libby, European Heart Journal 2001
Lumen
Thin fibrous cap
Collagen-poor

5. Degradadation fragments (3/4 & 1/4)
Small peptides / Amino Acids
Fibrillar collagen
MMP-13 / collagenase-3
MMP-8 / collagenase-2
MMP-1 / collagenase-1
MMP-13 / collagenase-3
MMP-2 / gelatinase-A
MMP-9 / gelatinase-B
Collagen breakdown by collagenases may
contribute to the vulnerability of atheroma

6. Lipid lowering by HMG-CoA reductase inhibitors reduces the
incidence of acute coronary events probably by stabilization
of “vulnerable” atherosclerotic plaques
Major statin trialsCholesterollevels
AverageHigh
Primary prevention Secondary prevention
WOSCOPS 4S
AFCAPS/TexCAPS
CARE
Pravastatin
24% risk reduction
Simvastatin
34% risk reduction
Lovastatin
40% risk reduction
Pravastatin
31% risk reduction
LIPID
Pravastatin
24% risk reduction
From Gotto and Farmer in Heart Disease (Braunwald, Zipes, Libby), 6th ed., 2001

7. Quantitative changes
Lesion size
Luminal area
Inward remodeling?
??
Atheromatous core
Macrophage-rich
Proteolytic activity (MMP)
Prothrombotic activity (TF)
Thin fibrous cap
Collagen-poor
Activated SMC
(MMP, TF)
Stabilization of “vulnerable”
plaques by lipid lowering
Lumen
“Regression”
“Stabilization”
Activated EC
VCAM-1, MCP-1
Masanori Aikawa and Peter Libby, European Heart Journal 2001
Qualitative or
functional changes
Macrophages
MMPs, TF
Collagen

8. Hypothesis 1
Do human and experimental
atherosclerotic plaques contain
collagenases?

9. Hypothesis 2
Does lipid lowering improve features
associated with plaque vulnerability:
(i.e, macrophage accumulation,
collagenase expression, collagen
content)?

10. Hypothesis 3
Does collagenolysis regulate
collagen accumulation of
atheroma in genetically-altered
mice?

11. Macrophage expression of collagenases
in human atheroma
Galis ZS, et al. JCI 1994 Sukhova GK, et al. Circulation 2000
MMP-1 / Collagenase-1 MMP-13 / Collagenase-3
Macrophages

12. Cell-typeMMP-8
EC
SMC
MØ
MMP-8/collagenase-2
expression by all cell
types in human
atheroma
Herman et al.
Circulation 2001

13. MMP-1/collagenase-1 expression in
atheroma of cholesterol-fed rabbits
Smooth muscle cells Macrophages MMP-1/collagenase-1
Masanori Aikawa, Elena Rabkin, Yoshikatsu Okada, Sami Voglic, Steven Clinton,
Constance Brinckerhoff, Galina Sukhova, Peter Libby Circulation 1998;97:2433-2444

14. MMP-13/collagenase-3 expression by
macrophages in atheroma
of apoE-deficient mice
MacrophagesMacrophages MMP-13 / collagenase-3MMP-13 / collagenase-3
Fukumoto, Libby, Rabkin, Deguchi, Varo, Harada, Hill, Lawler,
Schoen, Krane, Aikawa. Unpublished data.

15. MMP-3/stromelysin-1 MMP-9/gelatinase-B
MMP-3 and MMP-9 expression in atheroma
of Watanabe heritable hyperlipidemic
(WHHL) rabbits
Yoshihiro Fukumoto, Peter Libby, Elena Rabkin, Christopher C. Hill,
Makoto Enomoto, Yasuhiko Hirouchi, Masashi Shiomi,
Masanori Aikawa Circulation 2001;103:993-999

16. Dietary lipid lowering reduces
macrophages, MMP expression,
and increases collagen content
(a key determinant of plaque
stability) in atheroma of
cholesterol-fed rabbits
Masanori Aikawa, Elena Rabkin, Yoshikatsu Okada, Sami J. Voglic,
Steven K. Clinton, Constance E. Brinckerhoff, Galina K. Sukhova,
Peter Libby Circulation 1998;97:2433-2444
Decreased collagenase
and increased collagen

17. Dietary lipid lowering reduces macrophages
in atheroma of cholesterol-fed rabbits
Baseline lesion After lipid lowering
Masanori Aikawa, Elena Rabkin,
Yoshikatsu Okada, Sami Voglic,
Steven Clinton, Constance Brinckerhoff,
Galina Sukhova, Peter Libby
Circulation 1998;97:2433-2444

18. Collagen (Sirius red)
Masanori Aikawa,
Elena Rabkin,
Yoshikatsu Okada,
Sami J. Voglic,
Steven K. Clinton,
Constance E. Brinckerhoff,
Galina K. Sukhova,
Peter Libby
Circulation 1998;97:2433-2444
Dietary lipid lowering
reduces collagenase
expression and
increases collagen
accumulation
in atheroma of
cholesterol-fed
rabbits
Baseline lesion
Collagenase-1/MMP-1 Collagen
After Lipid Lowering

19. Diet lipid lowering promotes accumulation
of mature SMCs expressing less MMPs
in atheroma of cholesterol-fed rabbits
Masanori Aikawa, Elena Rabkin, Sami J. Voglic, Helen Shing, Ryozo Nagai, Frederick J. Schoen, Peter Libby
Circulation Research 1998;83:1015-1026

20. Control GroupControl Group
MMφφ MMP-1MMP-1
MMφφ MMP-1MMP-1
Cerivastatin GroupCerivastatin Group
Cerivastatin
reduces
macrophage
accumulation
and MMP
expression
in atheroma of
WHHL rabbits
Masanori Aikawa,
Elena Rabkin,
Seigo Sugiyama,
Sami J. Voglic,
Yoshihiro Fukumoto,
Yutaka Furukawa,
Masashi Shiomi,
Frederick J. Schoen,
Peter Libby
Circulation 2001;103:276-283
Plaque Stabilization by Statins 1
Effects on Evolution of Atheroma

21. Cerivastatin treatment reduces MMP expression
by macrophages in atheroma of WHHL rabbits
MMP-1/collagenase-1 MMP-9/gelatinase-BMMP-3/stromelysin-1
Masanori Aikawa, Elena Rabkin, Seigo Sugiyama, Sami Voglic, Yoshihiro Fukumoto, Yutaka Furukawa,
Masashi Shiomi, Frederick J. Schoen, Peter Libby Circulation 2001;103:276-283
CerivastatinControl
0
10
20
30
40
50 p<0.01
0
20
40
60
80
CerivastatinControl
0
10
20
30
40
p<0.01
CerivastatinControl
p<0.01
MMP-positivemacrophages(%)
n=9 n=10 n=9 n=10n=9 n=10
Plaque Stabilization by Statins 1
Effects on Evolution of Atheroma

22. CerivastatinCerivastatin
ControlControlCerivastatin increases
interstitial collagen
accumulation
in atheroma of
WHHL rabbits
Picrosirius red polarizationPicrosirius red polarization
Masanori Aikawa, Elena Rabkin,
Seigo Sugiyama, Sami J. Voglic,
Yoshihiro Fukumoto, Yutaka Furukawa,
Masashi Shiomi, Frederick J. Schoen,
Peter Libby
Circulation 2001;103:276-283
Plaque Stabilization by Statins 1
Effects on Evolution of Atheroma

23. Pravastatin (hydrophilic) and fluvastatin (lipophilic)
reduce MMP in macrophages in atheroma of WHHL
rabbits to a similar extent
MMP-1 (+) Mφ
100
Cont
n=10
Prava
n=9
Fluva
n=10
0
50
%
p<0.01
MMP-3 (+) Mφ
Cont
n=10
Prava
n=9
Fluva
n=10
0
50
100
%
p<0.05
p<0.05
100
MMP-9 (+) Mφ
Cont
n=10
Prava
n=9
Fluva
n=10
0
50
%
p<0.01
p<0.01
Yoshihiro Fukumoto, Peter Libby, Elena Rabkin, Christopher C. Hill, Makoto Enomoto,
Yasuhiko Hirouchi, Masashi Shiomi, Masanori Aikawa Circulation 2001;103:993-999
Plaque Stabilization by Statins 2
Differential Effects of Statins

24. Picrosirius Red Polarization
Atheroma of pravastatin
-treated WHHL rabbits
contain more interstitial
collagen than those
treated with fluvastatin
Yoshihiro Fukumoto, Peter Libby,
Elena Rabkin, Christopher C. Hill,
Makoto Enomoto, Yasuhiko Hirouchi,
Masashi Shiomi, Masanori Aikawa
Circulation 2001;103:993-999
Control
Pravastatin
Fluvastatin
Cont
n=10
Prava
n=9
Fluva
n=10
0
5
10
15 p<0.01 p<0.01
%-positivearea
Plaque Stabilization by Statins 2
Differential Effects of Statins

25. Pravastatin Fluvastatin
Procollagen-I
mRNA
SMα-actin
Pravastatin-treated WHHL rabbits contain
more procollagen-I mRNA expression than
those with fluvastatin treatment
Yoshihiro Fukumoto, Peter Libby, Elena Rabkin, Christopher C. Hill, Makoto Enomoto,
Yasuhiko Hirouchi, Masashi Shiomi, Masanori Aikawa Circulation 2001;103:993-999

26. Diet Statins
Lesion size………………………………...Decreased No change
Macrophage accumulation……………..Decreased Decreased
Macrophage proliferation………………. Decreased
Macrophage apoptosis…………………. No change
MMP expression/activity………………..Decreased Decreased
Collagen accumulation………………….Increased Increased
Tissue factor expression/activity……...Decreased Decreased
PAI-1 expression…………………………. Decreased
CD154 (CD40L) expression……………..Decreased Decreased
PDGF-B expression………………………Decreased
SMC differentiation……………………….Increased
Lipid lowering in rabbit atheroma

27. Lipid lowering in rabbit atheroma
Diet Statins
apoB-100 accumulation…………………Decreased
MDA accumulation……………………….Decreased Decreased
ROS production…………………………..Decreased
VCAM-1 expression……………………...Decreased
MCP-1 expression………………………..Decreased
eNOS expression………………………...Increased
Microvessels.……………………………..Decreased
Aikawa M, et al. Circulation 1998;97:2433-2444, Circ Res 1998;83:1015-1026,
Circulation 1999;100:1215-1222, Circulation 2001;103:276-283,
Circulation in revision
Fukumoto Y, et al. Circulation 2001;103:993-999, Circulation in revision
McConnell M, et al. Arterioscler Thromb Vasc Biol 1999;19:1956-1959

28. Collagen accumulation
MMP-collagenases (MMP-1, MMP-13)
Inverse relationship between collagenase
expression and collagen content in atheroma
during lipid lowering suggests the crucial
role of collagenase in collagen balance.
However, no direct evidence links
collagenases with regulation of the collagen
content of atheroma.
??

29. “Genetically-determined resistance to collagenase
action alters content of collagen and smooth
muscle cells in atheroma”
Brigham and Women’s Hospital, Massachusetts General Hospital,
Harvard Medical School, Boston, MA
Yoshihiro Fukumoto, Peter Libby, Elena Rabkin, Jun-o Deguchi,
Nerea Varo, Koichiro Harada, Christopher C. Hill, Patrick R. Lawler,
Frederick J. Schoen, Stephen M. Krane, Masanori Aikawa
ACC2002 Young Investigator Awards
Molecular and Cellular Cardiology
Monday, March 18, 2 PMMonday, March 18, 2 PM
Georgia World Congress Center, Room 257W
In vivo direct evidence of the role of collagenase

30. Fibrillar collagen
MMP-13 / collagenase-3
MMP-8 / collagenase-2
MMP-1 / collagenase-1
MMP-13 / collagenase-3
MMP-2 / gelatinase-A
MMP-9 / gelatinase-B
Collagenase-resistant mutant “knock-in” mouse
Mutation at specific cleavage
Site of type-I collagen
for MMP-collagenases

31. Does collagenase-resistance preserve more
collagen in mouse atheroma ?
Col+/+
/ apoE-/-
ColR/R
/ apoE-/-
??

32. Conclusion
Collagenases play a critical role in
collagen metabolism in atheroma.

33. Acknowledgment
Brigham and Women’s Hospital, Boston
Yoshihiro Fukumoto
Elena Rabkin
Christopher C. Hill
Sami J. Voglic
Seigo Sugiyama
Jun-o Deguchi
Koichiro Harada
Nerea Varo
Patrick Lawler
MGH, Boston
Steve M, Krane
Univ. of Tokyo
Ryozo Nagai
Kobe Univ.
Masashi Shiomi
USC, LA
Alex Sevanian
Yoshikatsu Okada
Yutaka Furukawa
Eugenia Shvartz
Helen Shing
Galina K. Sukhova
Michael Herman
Uwe Schoenbeck
Frederick J. Schoen