The balancee of proteolysis

Masanori Aikawa, MD, PhD Peter Libby, MD

Cardiovascular Medicine Brigham and Women’s Hospital

Harvard Medical SchoolBoston, MA

The balance of power in “vulnerable” plaques: collagenases vs. collagen

AbstractRupture 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.

M.L. Higuchi

ThrombusThrombus

Thin Thin Fibrous Fibrous CapCap

Lipid CoreLipid Core

Rupture of the “vulnerable” atherosclerotic Rupture of the “vulnerable” atherosclerotic plaque triggers acute coronary syndromes plaque triggers acute coronary syndromes

Atheromatous coreLipid, Macrophage-richProteolytic 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 capCollagen-poor

Degradadation fragments (3/4 & 1/4)

Small peptides / Amino Acids

Fibrillar collagen

MMP-13 / collagenase-3MMP-8 / collagenase-2MMP-1 / collagenase-1

MMP-13 / collagenase-3MMP-2 / gelatinase-AMMP-9 / gelatinase-B

Collagen breakdown by collagenases may contribute to the vulnerability of atheroma

Lipid lowering by HMG-CoA reductase inhibitors reduces the incidence of acute coronary events probably by stabilization of “vulnerable” atherosclerotic plaques

Major statin trialsCh

oles

tero

l lev

els

Ave

rage

High

Primary prevention Secondary prevention

WOSCOPS 4S

AFCAPS/TexCAPSCARE

Pravastatin24% risk reduction

Simvastatin34% risk reduction

Lovastatin40% risk reduction

Pravastatin31% risk reduction

LIPIDPravastatin

24% risk reduction

From Gotto and Farmer in Heart Disease (Braunwald, Zipes, Libby), 6th ed., 2001

Quantitative changes Lesion size Luminal area Inward remodeling?

??Atheromatous coreMacrophage-richProteolytic activity (MMP) Prothrombotic activity (TF)

Thin fibrous capCollagen-poorActivated SMC (MMP, TF)

Stabilization of “vulnerable” plaques by lipid lowering

Lumen

“Regression”

“Stabilization”Activated ECVCAM-1, MCP-1

Masanori Aikawa and Peter Libby, European Heart Journal 2001

Qualitative or functional changes Macrophages MMPs, TF Collagen

Hypothesis 1

Do human and experimental atherosclerotic plaques contain collagenases?

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

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

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

Cell-typeMMP-8

EC

SMC

MØ

MMP-8/collagenase-2 expression by all cell types in human atheromaHerman et al.

Circulation 2001

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

MMP-13/collagenase-3 expression by macrophages in atheroma

of apoE-deficient miceMacrophagesMacrophages MMP-13 / collagenase-3MMP-13 / collagenase-3

Fukumoto, Libby, Rabkin, Deguchi, Varo, Harada, Hill, Lawler, Schoen, Krane, Aikawa. Unpublished data.

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

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

Dietary lipid lowering reduces macrophagesin 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

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 loweringreduces collagenase expression and increases collagenaccumulation in atheroma of cholesterol-fed rabbits

Baseline lesion

Collagenase-1/MMP-1 Collagen

After Lipid Lowering

Diet lipid lowering promotes accumulation of mature SMCs expressing less MMPsin atheroma of cholesterol-fed rabbits

Masanori Aikawa, Elena Rabkin, Sami J. Voglic, Helen Shing, Ryozo Nagai, Frederick J. Schoen, Peter LibbyCirculation Research 1998;83:1015-1026

Control GroupControl Group

MM MMP-1MMP-1

MM 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 LibbyCirculation 2001;103:276-283

Plaque Stabilization by Statins 1Effects on Evolution of Atheroma

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

CerivastatinControl0

10

20

30

40

50 p<0.01

0

20

40

60

80

CerivastatinControl0

10

20

30

40 p<0.01

CerivastatinControl

p<0.01

MM

P-po

sitiv

e m

acro

phag

es (%

)

n=9 n=10 n=9 n=10n=9 n=10

Plaque Stabilization by Statins 1Effects on Evolution of Atheroma

CerivastatinCerivastatin

ControlControlCerivastatin increasesinterstitial collagen accumulationin atheroma of WHHL rabbitsPicrosirius red polarizationPicrosirius red polarizationMasanori 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 1Effects on Evolution of Atheroma

Pravastatin (hydrophilic) and fluvastatin (lipophilic) reduce MMP in macrophages in atheroma of WHHL rabbits t

o a similar extentMMP-1 (+) M

100

Contn=10

Pravan=9

Fluvan=10

0

50

%p<0.01

MMP-3 (+) M

Contn=10

Pravan=9

Fluvan=10

0

50

100%

p<0.05p<0.05

100

MMP-9 (+) M

Contn=10

Pravan=9

Fluvan=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 2Differential Effects of Statins

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

FluvastatinContn=10

Pravan=9

Fluvan=10

05

1015 p<0.01 p<0.01

%-p

ositi

ve a

rea

Plaque Stabilization by Statins 2Differential Effects of Statins

Pravastatin Fluvastatin

Procollagen-I mRNA

SM-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

Diet StatinsLesion size………………………………...Decreased No changeMacrophage accumulation……………..Decreased Decreased Macrophage proliferation………………. Decreased Macrophage apoptosis…………………. No changeMMP expression/activity………………..Decreased DecreasedCollagen accumulation………………….Increased IncreasedTissue factor expression/activity……...Decreased DecreasedPAI-1 expression…………………………. DecreasedCD154 (CD40L) expression……………..Decreased DecreasedPDGF-B expression………………………DecreasedSMC differentiation……………………….Increased

Lipid lowering in rabbit atheroma

Lipid lowering in rabbit atheromaDiet Statins

apoB-100 accumulation…………………DecreasedMDA accumulation……………………….Decreased DecreasedROS production…………………………..DecreasedVCAM-1 expression……………………...DecreasedMCP-1 expression………………………..DecreasedeNOS expression………………………...IncreasedMicrovessels.……………………………..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 revisionMcConnell M, et al. Arterioscler Thromb Vasc Biol 1999;19:1956-1959

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.

??

“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 AwardsMolecular and Cellular Cardiology

Monday, March 18, 2 PM Monday, March 18, 2 PM Georgia World Congress Center, Room 257W

In vivo direct evidence of the role of collagenase

Fibrillar collagen

MMP-13 / collagenase-3MMP-8 / collagenase-2MMP-1 / collagenase-1

MMP-13 / collagenase-3MMP-2 / gelatinase-AMMP-9 / gelatinase-B

Collagenase-resistant mutant “knock-in” mouse

Mutation at specific cleavageSite of type-I collagen for MMP-collagenases

Does collagenase-resistance preserve more collagen in mouse atheroma ?

Col+/+ / apoE-/- ColR/R / apoE-/-

??

Conclusion

Collagenases play a critical role in collagen metabolism in atheroma.

AcknowledgmentBrigham and Women’s Hospital, BostonYoshihiro Fukumoto Elena Rabkin Christopher C. HillSami J. VoglicSeigo SugiyamaJun-o DeguchiKoichiro HaradaNerea VaroPatrick Lawler

MGH, BostonSteve M, Krane

Univ. of TokyoRyozo NagaiKobe Univ.Masashi ShiomiUSC, LAAlex Sevanian

Yoshikatsu OkadaYutaka FurukawaEugenia ShvartzHelen Shing Galina K. SukhovaMichael HermanUwe SchoenbeckFrederick J. Schoen