Macrophage induced proteolysis zorina galis- emory u

Content and GraphicsContent and Graphics
Zorina Galis, Ph.D.Zorina Galis, Ph.D.
“Macrophage-induced proteolysis:
how many MMPs and non-MMPs are
involved?”?
Zorina S. Galis, Ph.D.Zorina S. Galis, Ph.D.
Division of Cardiology , Emory UniversityDivision of Cardiology , Emory University
School of MedicineSchool of Medicine
Department of Biomedical EngineeringDepartment of Biomedical Engineering
Emory/Georgia Tech,Emory/Georgia Tech, Atlanta GAAtlanta GA
March 16, 2002March 16, 2002
33rdrd
Vulnerable Plaque SymposiumVulnerable Plaque Symposium
March 16, 2002March 16, 2002
33rdrd
Vulnerable Plaque SymposiumVulnerable Plaque Symposium

LipidLipid
corecore
ThrombusThrombus
Natural history of human atherosclerosisNatural history of human atherosclerosisNatural history of human atherosclerosisNatural history of human atherosclerosis
M. Davies, 1998M. Davies, 1998
Acute cardiovascular events representAcute cardiovascular events represent
a late stage of arterial remodelinga late stage of arterial remodeling
Acute cardiovascular events representAcute cardiovascular events represent
a late stage of arterial remodelinga late stage of arterial remodeling
adaptationadaptation
sustainedsustained
adaptationadaptation
and repairand repair
destructiondestruction
Culprit = ruptureCulprit = ruptureCulprit = ruptureCulprit = rupture

Selected MMPSelected MMP Selected substratesSelected substrates
StromelysinStromelysin
(SL / MMP-3)(SL / MMP-3)
StromelysinStromelysin
(SL / MMP-3)(SL / MMP-3)
Proteoglycans, fibronectin, lamininProteoglycans, fibronectin, laminin
pro-MMP-1, pro-MMP-9pro-MMP-1, pro-MMP-9
Proteoglycans, fibronectin, lamininProteoglycans, fibronectin, laminin
pro-MMP-1, pro-MMP-9pro-MMP-1, pro-MMP-9
Gelatinases (GL)Gelatinases (GL)
72 kD GL, GL a (MMP-2)72 kD GL, GL a (MMP-2)
92 kD GL, GL b (MMP-9)92 kD GL, GL b (MMP-9)
Gelatinases (GL)Gelatinases (GL)
72 kD GL, GL a (MMP-2)72 kD GL, GL a (MMP-2)
92 kD GL, GL b (MMP-9)92 kD GL, GL b (MMP-9)
Collagen type IV / VCollagen type IV / V
degraded collagen, elastindegraded collagen, elastin
Collagen type IV / VCollagen type IV / V
degraded collagen, elastindegraded collagen, elastin
Interstitial collagenaseInterstitial collagenase
(CL / MMP-1)(CL / MMP-1)
Interstitial collagenaseInterstitial collagenase
(CL / MMP-1)(CL / MMP-1) Fibrillar collagenFibrillar collagenFibrillar collagenFibrillar collagen
membrane-type MMP-1membrane-type MMP-1
(MT-MMP)(MT-MMP)
membrane-type MMP-1membrane-type MMP-1
(MT-MMP)(MT-MMP)
pro-MMP-2, pro-MMP-13,pro-MMP-2, pro-MMP-13,
collagen, fibronectin, laminincollagen, fibronectin, laminin
pro-MMP-2, pro-MMP-13,pro-MMP-2, pro-MMP-13,
collagen, fibronectin, laminincollagen, fibronectin, laminin
The matrix metalloproteinase (MMP) family ofThe matrix metalloproteinase (MMP) family of
enzymes can break-down matrix componentsenzymes can break-down matrix components
The matrix metalloproteinase (MMP) family ofThe matrix metalloproteinase (MMP) family of
enzymes can break-down matrix componentsenzymes can break-down matrix components
MatrilysinMatrilysin
(MMP-7)(MMP-7)
MatrilysinMatrilysin
(MMP-7)(MMP-7)
fibronectin, collagen type IV,fibronectin, collagen type IV,
laminin, elastinlaminin, elastin
fibronectin, collagen type IV,fibronectin, collagen type IV,
laminin, elastinlaminin, elastin
Could MMPs beCould MMPs be
responsible for theresponsible for the
weakening ofweakening of
atherosclerotic plaques?atherosclerotic plaques?

Galis et al. 1994, JCIGalis et al. 1994, JCI
Normal coronary arteryNormal coronary artery Coronary atheromaCoronary atheroma
Immunohistochemistry: MMP-3Immunohistochemistry: MMP-3 Immuhistochemistry:Immuhistochemistry: MMP-3MMP-3
LumenLumen
fibrous capfibrous cap
In situIn situ zymography (activity assay)zymography (activity assay)In situIn situ zymography (activity assay)zymography (activity assay)
Lysis ofLysis of
fluorescentfluorescent
substratesubstrate
First…First…
are MMPs expressed in human atheroma?are MMPs expressed in human atheroma?
First…First…
are MMPs expressed in human atheroma?are MMPs expressed in human atheroma?
The shoulders of human atherosclerotic
plaques contain active MMPs
MMP proteins are overexpressed in
the vulnerable shoulders, but are they
enzymaticaly active ?
MMP proteins are overexpressed in
the vulnerable shoulders, but are they
enzymaticaly active ?

Vulnerable plaques have a high percentage of
macrophage-foam cells
Farb &VirmaniFarb &VirmaniFarb &VirmaniFarb &Virmani
LumenLumenMacrophageMacrophage
foam cellsfoam cells

Macrophage (MΦ) foam cells in the shoulders
of human atheroma express MMPs
Immunohistology: Detection of MMP-3Immunohistology: Detection of MMP-3 Double immunohistology:
Detection of MMP-1 and MΦ
Double immunohistology:
Detection of MMP-1 and MΦ
(Galis et al., 1994, J Clin Invest)(Galis et al., 1994, J Clin Invest)

Other MMP sightings in the
atheroma…
Other MMP sightings in the
atheroma…
• Messenger RNA for MMP-3 colocalizes with macrophageMessenger RNA for MMP-3 colocalizes with macrophage
foam cells (Henney et al., 1991)foam cells (Henney et al., 1991)
• Active MMP-9 synthesis in atherectomy specimens fromActive MMP-9 synthesis in atherectomy specimens from
patients with unstable angina and MMP-13 colocalizepatients with unstable angina and MMP-13 colocalize
with degraded collagen (Brown et al, 1995)with degraded collagen (Brown et al, 1995)
• MMP-7 is expressed by macrophage foam cells at sitesMMP-7 is expressed by macrophage foam cells at sites
of potential plaque rupture (Halpert et al. 1996)of potential plaque rupture (Halpert et al. 1996)
• Macrophages can also express the elastolytic MMP-8Macrophages can also express the elastolytic MMP-8
(Herman, 2001)(Herman, 2001)
Macrophage foam cells are associated with
increased MMP expression and activity
Macrophage foam cells are associated with
increased MMP expression and activity

Do MMPs degrade the collagen of the
fibrous cap?
Do MMPs degrade the collagen of the
fibrous cap?
• Macrophage MMP-2 and MMP-9 degrade ex vivoMacrophage MMP-2 and MMP-9 degrade ex vivo
the collagen of the plaque’s fibrous cap (Shah etthe collagen of the plaque’s fibrous cap (Shah et
al, 1995)al, 1995)
• MMP-1 and MMP-13 colocalize in the plaque withMMP-1 and MMP-13 colocalize in the plaque with
epitopes expressed by degraded collagenepitopes expressed by degraded collagen
(immunohistochemistry, Sukhova et al, 1999)(immunohistochemistry, Sukhova et al, 1999)
• MMP-8 colocalizes with epitopes expressed byMMP-8 colocalizes with epitopes expressed by
cleaved type I collagen in the shoulders ofcleaved type I collagen in the shoulders of
human plaque (immunohistochemistry, Hermanhuman plaque (immunohistochemistry, Herman
et al., 2001)et al., 2001)

Overexpression of interstitialOverexpression of interstitial
collagenase (MMP-1 ) coincidescollagenase (MMP-1 ) coincides
with the places subjected to thewith the places subjected to the
highest tensile stress within thehighest tensile stress within the
vulnerable shoulders (Leevulnerable shoulders (Lee et alet al..
1996)1996)
Bad luck?!?Bad luck?!?Bad luck?!?Bad luck?!?

Determinants of atherosclerotic plaque stabilityDeterminants of atherosclerotic plaque stability
Tissue characteristicsTissue characteristics Mechanical stressMechanical stress
Active degradation of matrixActive degradation of matrix
scaffold in the vulnerablescaffold in the vulnerable
shoulders by MMPsshoulders by MMPs
(Galis(Galis et alet al 1994, Galis1994, Galis et al.et al. 1995)1995)
Tissue characteristicsTissue characteristics Mechanical stressMechanical stress
Rupture of atherosclerotic plaqueRupture of atherosclerotic plaqueRupture of atherosclerotic plaqueRupture of atherosclerotic plaque
thin fibrous capthin fibrous cap
large lipid corelarge lipid core
((Cheng et al. 1993)Cheng et al. 1993)
inflammationinflammation
((LendonLendon et al.et al. 1991,1991,
van der Walvan der Wal et alet al.,.,
1994)1994)
mechanical “hotmechanical “hot
spots”coincide with thespots”coincide with the
weak pointsweak points
((Lee et al. 1996)Lee et al. 1996)

• Cytokine stimulation: human EC (Hanemaaijeret al. 1993), human SMC (Galis et
al. 1994)
• mechanical stretch: shoulders (Lee et al, 1996)
• engagement of cell surface receptors: VCAM-1 (Romanic & Madri 1994), CD40
(Malik 1996, Schonbeck, Mach et al 1997), ICAM-1 (Aoudjit et al 1998)
• modified lipoproteins: vascular cells (Rajavashist et al. 1999), macrophages
(Xu et al 1999)
• proteases: thrombin (Galis et al. 1995), plasmin/uPA (Carmeliet et al. 1997),
cathepsins (Sukhova et al 1997), MT-MMP (Wang et al.,1998)
• oxidative stress:
• increased by superoxide, hydrogen peroxide, peroxynitrite (Rajagopalan et al. 1996),
• Inhibited by N-acetyl cysteine (Galis et al. 1998), nitric oxide (Gurjar et al. 1999)
• matrix composition: collagen I increases macrophage MMP(Wesley et al.
1997)
• Infections: Chlamydia (Kol et al, 1998)Kol et al, 1998)
• growth factors: FGF-2 (Pickering et al 1997); VEGF (Wang& Keiser 1998)
Potential modulators of MMP expression
and activity in atheroma
Expression of pro-MMPs
Activation of MMP enzymatic activity

Experimental modelExperimental model
for investigation offor investigation of
macrophage foam cellmacrophage foam cell
MMPsMMPs
Subcutaneous granulomaSubcutaneous granuloma
Balloon angioplastyBalloon angioplasty
HypercholesterolemicHypercholesterolemic
dietdiet
MacrophageMacrophage
(anti-RAM 11)(anti-RAM 11)
Intracellular
lipid
(Nile red)

0
200
400
600
MΦ FC
x103
counts/min/106
cells
PMA - + - +
Macrophage (MMacrophage (MΦΦ)-derived)-derived
foam cells (FC) producefoam cells (FC) produce
reactive oxygen speciesreactive oxygen species
(Rajagopalan et al. 1996 JCI)(Rajagopalan et al. 1996 JCI)
Macrophage-derived FCMacrophage-derived FC
activate the zymogen ofactivate the zymogen of
MMP-9MMP-9 in vitroin vitro
(Galis et al., 1998 Circulation)(Galis et al., 1998 Circulation)
Macrophage-derived FCMacrophage-derived FC
activate the zymogen ofactivate the zymogen of
MMP-9MMP-9 in vitroin vitro
SuperoxideSuperoxide
PeroxidesPeroxides
MΦMΦ FCFC
66 -66 -
97 -97 -
46 -46 -
pro-MMP-9pro-MMP-9
MMP-9MMP-9
MMΦΦ FCFC
MMP-9MMP-9

Reactive oxygen species activate latent MMPsReactive oxygen species activate latent MMPs
produced by human vascular smooth muscle cellsproduced by human vascular smooth muscle cells
++
Pro-MMP-2
Pro-MMP-9
MMP-9
MMP-2
Pro-MMP-2
MMP-2
In vivo?In vivo?In vivo?In vivo?
+ Xanthine/
Xanthine Oxidase
+ Xanthine/
Xanthine Oxidase
(Rajagopalan et al. 1996, JCI)(Rajagopalan et al. 1996, JCI)

N-acetyl cysteine (NAC) treatment decreasesN-acetyl cysteine (NAC) treatment decreases in situin situ
MMP-9 expression and activity in experimental rabbitMMP-9 expression and activity in experimental rabbit
atheromaatheroma
MMP-9 Macrophages
MMP-9
+ NAC
Macrophages
+ NAC100100 µµmm
IELIEL
Pro-MMP-2 -
MWM
(kDa)
Abdominal
aorta
Thoracic
aorta
+ NAC + NAC0 0
MMP-2 -
Pro-MMP-9 -
MMP-9 -

Lipid lowering therapy may increase
plaque stability by decreasing the
oxidative stress
•improvement of endothelialimprovement of endothelial
functionfunction
•decreased plaque lipiddecreased plaque lipid
•decreased MMP productiondecreased MMP production
increased plaque stabilityincreased plaque stability
•improvement of endothelialimprovement of endothelial
functionfunction
•decreased MMP productiondecreased MMP production
increased plaque stabilityincreased plaque stability
OxidativeOxidative
stressstress
LipidLipid
Therapeutic interventions and plaque stabilityTherapeutic interventions and plaque stabilityTherapeutic interventions and plaque stabilityTherapeutic interventions and plaque stability

Experimental macrophage-rich arterial lesions
(ApoE KO mouse carotid artery ligation)
Macrophage-Macrophage-
rich neointimarich neointima
Normal carotid arteryNormal carotid arteryNormal carotid arteryNormal carotid artery Atherosclerotic carotid arteryAtherosclerotic carotid arteryAtherosclerotic carotid arteryAtherosclerotic carotid artery
(Lessner et al., unpublished)(Lessner et al., unpublished)

Our studies indicate that arteries withOur studies indicate that arteries with
macrophage-rich lesions undergomacrophage-rich lesions undergo
enhanced positive remodelingenhanced positive remodeling
Our studies indicate that arteries withOur studies indicate that arteries with
macrophage-rich lesions undergomacrophage-rich lesions undergo
enhanced positive remodelingenhanced positive remodeling
55 1010 1515 2020 2525 303055 1010 1515 2020 2525 3030
Macrophage areaMacrophage area
10001000
20002000
Outer perimeterOuter perimeter
Time
(days)
Time
(days)
1010
2020
3030
4040
AreaArea
((µµmm22
)) LengthLength
((µµm)m)
- -
Pro MMP-9
Pro MMP-2
98 kDa
72 kDa
- -
Days 0 14 28
WT WT WTKO KO KOST
Arteries with macrophage-rich lesions have
enhanced MMP activity
Arteries with macrophage-rich lesions have
enhanced MMP activity

Positively remodeling isPositively remodeling is
associated with plaque instabilityassociated with plaque instability
Positively remodeling isPositively remodeling is
associated with plaque instabilityassociated with plaque instability
Schoenhagen et al., 2000 Circulation 101Schoenhagen et al., 2000 Circulation 101Schoenhagen et al., 2000 Circulation 101Schoenhagen et al., 2000 Circulation 101
UnstableUnstable
StableStable
PositivePositive NegativeNegativeAbsentAbsent
2020
3030
4040
5050
1010
RemodelingRemodeling
%cohort%cohort

FoamFoam
cell/macrophage-cell/macrophage-
drivendriven
Smooth muscleSmooth muscle
cell-drivencell-driven
UnstableUnstable
plaqueplaque
StableStable
plaqueplaque
Constrictive arterial remodelingConstrictive arterial remodeling
Outward arterial remodelingOutward arterial remodeling(Galis and(Galis and
Khatri, 2002)Khatri, 2002)
Normal arteryNormal artery

Macrophage-foam cells are key regulators of
MMP-dependent degradation of vascular
matrix
Macrophage-foam cells are key regulators of
MMP-dependent degradation of vascular
matrix
ROSROS
TFTF
ThrombinThrombin
ModulateModulate
MMPMMP
activityactivity
ExpressExpress
pro-pro-
MMPsMMPs
CytokinesCytokines
UpregulateUpregulate
vascular cellvascular cell
MMPsMMPs
Macrophage foam cellsMacrophage foam cells

Macrophage non-MMP proteases?Macrophage non-MMP proteases?Macrophage non-MMP proteases?Macrophage non-MMP proteases?
• CathepsinsCathepsins K and S
– have elastolytic activityhave elastolytic activity
– Expressed by macrophages in atheroma (Sukhova etExpressed by macrophages in atheroma (Sukhova et
al. 1998)al. 1998)
• ThrombinThrombin
– generated at sites of disruption, can be generated ingenerated at sites of disruption, can be generated in
the atherosclerotic plaques via tissue factorthe atherosclerotic plaques via tissue factor
expressed by macrophage foam cells (Wilcox et al.expressed by macrophage foam cells (Wilcox et al.
1989)1989)
– can activate latent MMP-2 (Galis et al., 1997)can activate latent MMP-2 (Galis et al., 1997)

Potential protease networksPotential protease networksPotential protease networksPotential protease networks
• Macrophage MMPs may increase the activity of otherMacrophage MMPs may increase the activity of other
proteases through:proteases through:
– Activation of MMP zymogens - e.g., MMP-3 can activate pro-Activation of MMP zymogens - e.g., MMP-3 can activate pro-
MMP-1MMP-1
– inactivation of inhibitors - e.g., MMP-1, MMP-3, and MMP-9caninactivation of inhibitors - e.g., MMP-1, MMP-3, and MMP-9can
inactivate alpha 1-antitrypsin, the primary physiologic inhibitorinactivate alpha 1-antitrypsin, the primary physiologic inhibitor
of human leukocyte elastase (Sires et al. 1994of human leukocyte elastase (Sires et al. 1994); MMP-1, MMP-7,); MMP-1, MMP-7,
MMP-9, and MMP-12 cleave tissue factor pathway inhibitor (BelaaouajMMP-9, and MMP-12 cleave tissue factor pathway inhibitor (Belaaouaj
et al, 2000)et al, 2000)
• Macrophage non-MMPs may increase the activity ofMacrophage non-MMPs may increase the activity of
other proteases through:other proteases through:
– Activation of MMP zymogens -- e.g., uPA can activate pro-MMP-3,Activation of MMP zymogens -- e.g., uPA can activate pro-MMP-3,
thrombin can activate pro-MMP-2thrombin can activate pro-MMP-2
– inactivation of inhibitors ?inactivation of inhibitors ?

RupturedRuptured
plaqueplaque
The mutual stimulation of generation of activeThe mutual stimulation of generation of active
MMPs and thrombin may the basis of sustainedMMPs and thrombin may the basis of sustained
plaque instability, with recurring episodes ofplaque instability, with recurring episodes of
plaque disruption and thrombosisplaque disruption and thrombosis
MMPsMMPs
ThrombinThrombin
MMPsMMPs
ThrombinThrombin
ThrombinThrombin
activatesactivates
latent MMPslatent MMPs
(Galis et al.,(Galis et al.,
1997)1997)
Active MMPsActive MMPs
stimulatestimulate
generation ofgeneration of
thrombinthrombin
(Sawicki et(Sawicki et
al., 1997)al., 1997)
Proteases and the unstable atherosclerotic plaqueProteases and the unstable atherosclerotic plaqueProteases and the unstable atherosclerotic plaqueProteases and the unstable atherosclerotic plaque

Are these proteases redundant?Are these proteases redundant?
What are the potential actions of allWhat are the potential actions of all
these in relation to plaque rupture?these in relation to plaque rupture?
Are these proteases redundant?Are these proteases redundant?
What are the potential actions of allWhat are the potential actions of all
these in relation to plaque rupture?these in relation to plaque rupture?
• MMPs can degrade all the components of theMMPs can degrade all the components of the
extracellular matrixextracellular matrix
• uPAuPA activatesactivates MMP zymogens (MMP zymogens (Carmeliet et al. 1997)
• Thrombin can activate MMP zymogens (Galis et al. 1997)Thrombin can activate MMP zymogens (Galis et al. 1997)
Do MMPs provide a common pathway forDo MMPs provide a common pathway for
plaque weakening by other proteases?!?plaque weakening by other proteases?!?
Do MMPs provide a common pathway forDo MMPs provide a common pathway for
plaque weakening by other proteases?!?plaque weakening by other proteases?!?

ConclusionsConclusionsConclusionsConclusions
• Macrophages provide several essential components thatMacrophages provide several essential components that
can increase proteolytic activity within atheroscleroticcan increase proteolytic activity within atherosclerotic
plaquesplaques
– Produce proteases: MMPs, cathepsins, thrombin via TFProduce proteases: MMPs, cathepsins, thrombin via TF
– Provide activators for proteases: ROS, MMPsProvide activators for proteases: ROS, MMPs
– Provide means to inactivate protease inhibitors: MMPsProvide means to inactivate protease inhibitors: MMPs
– Stimulate production of proteases in vascular cellsStimulate production of proteases in vascular cells
All circumstantial evidence supports degradation of matrixAll circumstantial evidence supports degradation of matrix
due to an increased macrophage-related proteolytic activitydue to an increased macrophage-related proteolytic activity
as a mechanism thorough which macrophage infiltratesas a mechanism thorough which macrophage infiltrates
precipitate plaque destabilization, however…..precipitate plaque destabilization, however…..
All circumstantial evidence supports degradation of matrixAll circumstantial evidence supports degradation of matrix
due to an increased macrophage-related proteolytic activitydue to an increased macrophage-related proteolytic activity
as a mechanism thorough which macrophage infiltratesas a mechanism thorough which macrophage infiltrates
precipitate plaque destabilization, however…..precipitate plaque destabilization, however…..
The direct evidence is still missing!The direct evidence is still missing!The direct evidence is still missing!The direct evidence is still missing!

Macrophage induced proteolysis zorina galis- emory u

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