Vp 2002 talk show copy galis

Content and Graphics Content 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 University Division of Cardiology , Emory University School of MedicineSchool of Medicine

Department of Biomedical Engineering Department of Biomedical Engineering Emory/Georgia Tech, Emory/Georgia Tech, Atlanta GAAtlanta GA

March 16, 2002 March 16, 2002 33rdrd Vulnerable Plaque Symposium Vulnerable Plaque Symposium

http://www.emory.edu/WHSC/MED/med.html


Lipid Lipid corecore

ThrombusThrombus

Natural history of human atherosclerosisNatural history of human atherosclerosis

M. Davies, 1998M. Davies, 1998

Acute cardiovascular events represent Acute cardiovascular events represent a late stage of arterial remodelinga late stage of arterial remodeling

adaptationadaptationsustained sustained adaptation adaptation and repairand repair

destructiondestruction

Culprit = ruptureCulprit = rupture


Selected MMPSelected MMP Selected substratesSelected substrates

Stromelysin Stromelysin (SL / MMP-3)(SL / MMP-3)

Proteoglycans, fibronectin, lamininProteoglycans, fibronectin, lamininpro-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)

Collagen type IV / VCollagen type IV / Vdegraded collagen, elastindegraded collagen, elastin

Interstitial collagenaseInterstitial collagenase(CL / MMP-1)(CL / MMP-1) Fibrillar collagenFibrillar collagen

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

The matrix metalloproteinase (MMP) family of The matrix metalloproteinase (MMP) family of enzymes can break-down matrix componentsenzymes can break-down matrix components

MatrilysinMatrilysin(MMP-7)(MMP-7)

fibronectin, collagen type IV, fibronectin, collagen type IV, laminin, elastinlaminin, elastin

Could MMPs be Could MMPs be responsible for the responsible for the

weakening of weakening 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-3 MMP-3

LumenLumen

fibrous capfibrous cap

In situIn situ zymography (activity assay) zymography (activity assay)

Lysis of Lysis of fluorescent fluorescent substratesubstrate

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 ?


Vulnerable plaques have a high percentage of macrophage-foam cells

Farb &VirmaniFarb &Virmani

LumenLumenMacrophage Macrophage foam cellsfoam cells


Macrophage (M) foam cells in the shoulders of human atheroma express MMPs

Immunohistology: Detection of MMP-3 Double immunohistology: Detection of MMP-1 and M

(Galis et al., 1994, J Clin Invest)


Other MMP sightings in the atheroma…

• Messenger RNA for MMP-3 colocalizes with macrophage Messenger 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 from Active MMP-9 synthesis in atherectomy specimens from patients with unstable angina and MMP-13 colocalize patients 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 sites MMP-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-8 Macrophages can also express the elastolytic MMP-8 (Herman, 2001)(Herman, 2001)

Macrophage foam cells are associated with increased MMP expression and activity


Do MMPs degrade the collagen of the fibrous cap?

• Macrophage MMP-2 and MMP-9 degrade ex vivo Macrophage MMP-2 and MMP-9 degrade ex vivo the collagen of the plaque’s fibrous cap (Shah et the collagen of the plaque’s fibrous cap (Shah et al, 1995)al, 1995)

• MMP-1 and MMP-13 colocalize in the plaque with MMP-1 and MMP-13 colocalize in the plaque with epitopes expressed by degraded collagen epitopes expressed by degraded collagen (immunohistochemistry, Sukhova et al, 1999)(immunohistochemistry, Sukhova et al, 1999)

• MMP-8 colocalizes with epitopes expressed by MMP-8 colocalizes with epitopes expressed by cleaved type I collagen in the shoulders of cleaved type I collagen in the shoulders of human plaque (immunohistochemistry, Herman human plaque (immunohistochemistry, Herman et al., 2001)et al., 2001)


Overexpression of interstitial Overexpression of interstitial collagenase (MMP-1 ) coincides collagenase (MMP-1 ) coincides with the places subjected to the with the places subjected to the highest tensile stress within the highest tensile stress within the vulnerable shoulders (Lee vulnerable shoulders (Lee et alet al. . 1996)1996)

Bad luck?!?Bad luck?!?


Determinants of atherosclerotic plaque stabilityDeterminants of atherosclerotic plaque stabilityTissue characteristicsTissue characteristics Mechanical stress Mechanical stress

Active degradation of matrix Active degradation of matrix scaffold in the vulnerable scaffold in the vulnerable shoulders by MMPs shoulders by MMPs (Galis (Galis et alet al 1994, Galis 1994, Galis et al. et al. 1995)1995)

Tissue characteristicsTissue characteristics Mechanical stressMechanical stress

Rupture of atherosclerotic plaqueRupture of atherosclerotic plaque

thin fibrous capthin fibrous caplarge lipid corelarge lipid core ((Cheng et al. 1993)Cheng et al. 1993)

inflammationinflammation((Lendon Lendon et al.et al. 1991, van 1991, van der Wal der Wal et alet al., 1994)., 1994)

mechanical “hot mechanical “hot spots”coincide with the spots”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-MMPsActivation of MMP enzymatic activity


Experimental modelExperimental modelfor investigation of for investigation of

macrophage foam cell macrophage foam cell MMPsMMPs

Subcutaneous granulomaSubcutaneous granuloma

Balloon angioplastyBalloon angioplasty

Hypercholesterolemic Hypercholesterolemic dietdiet

MacrophageMacrophage(anti-RAM 11)(anti-RAM 11)

Intracellular lipid (Nile red)


0

200

400

600

M FC

x 10

3 cou

nts/

min

/106 c

ells

PMA - + - +

Macrophage (MMacrophage (M)-derived )-derived foam cells (FC) produce foam cells (FC) produce

reactive oxygen speciesreactive oxygen species(Rajagopalan et al. 1996 JCI)(Rajagopalan et al. 1996 JCI)

Macrophage-derived FC Macrophage-derived FC activate the zymogen of activate the zymogen of

MMP-9 MMP-9 in vitroin vitro(Galis et al., 1998 Circulation)(Galis et al., 1998 Circulation)

Superoxide

Peroxides

M FC

66 -66 -

97 -97 -

46 -46 -

pro-MMP-9pro-MMP-9

MMP-9MMP-9

MM FCFC

MMP-9


Reactive oxygen species activate latent MMPs Reactive 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-9MMP-2

Pro-MMP-2MMP-2

In vivo?In vivo?

+ Xanthine/ Xanthine Oxidase

(Rajagopalan et al. 1996, JCI)(Rajagopalan et al. 1996, JCI)


N-acetyl cysteine (NAC) treatment decreases N-acetyl cysteine (NAC) treatment decreases in situ in situ MMP-9 expression and activity in experimental rabbit MMP-9 expression and activity in experimental rabbit

atheroma atheroma MMP-9 Macrophages

MMP-9

+ NAC

Macrophages

+ NAC100 100 mm

IELIEL

Pro-MMP-2 -

MWM(kDa)

Abdominal aorta

Thoracic aorta

+ NAC + NAC0 0

MMP-2 -

Pro-MMP-9 -MMP-9 -

(Galis et al., 1998 Circulation)(Galis et al., 1998 Circulation)


Lipid lowering therapy may increase plaque stability by decreasing the

oxidative stress

•improvement of endothelial improvement of endothelial functionfunction•decreased plaque lipiddecreased plaque lipid•decreased MMP productiondecreased MMP productionincreased plaque stabilityincreased plaque stability

•improvement of endothelial improvement of endothelial functionfunction•decreased MMP productiondecreased MMP productionincreased plaque stabilityincreased plaque stability

Oxidative Oxidative stressstress

LipidLipid

Therapeutic 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 artery Atherosclerotic carotid arteryAtherosclerotic carotid artery

(Lessner et al., unpublished)(Lessner et al., unpublished)


Our studies indicate that arteries with Our studies indicate that arteries with macrophage-rich lesions undergo macrophage-rich lesions undergo

enhanced positive remodelingenhanced positive remodeling

55 1010 1515 2020 2525 303055 1010 1515 2020 2525 3030

Macrophage area

10001000

20002000

Outer perimeter

Time (days)

Time (days)

1010

2020

30304040

Area Area ((mm22)) Length Length

((m)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


Positively remodeling is Positively remodeling is associated with plaque instabilityassociated with plaque instability

Schoenhagen et al., 2000 Circulation 101Schoenhagen et al., 2000 Circulation 101

UnstableUnstable

StableStable

PositivePositive NegativeNegativeAbsentAbsent

2020

3030

4040

5050

1010

RemodelingRemodeling

% c

ohor

t%

coh

ort


Foam Foam cell/macrophage-cell/macrophage-drivendriven

Smooth muscle Smooth muscle cell-drivencell-driven

Unstable Unstable plaqueplaque

Stable Stable plaqueplaque

Constrictive arterial remodeling

Outward 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

ROS ROS TF TF

Thrombin Thrombin

Modulate Modulate MMP MMP

activityactivity

Express Express pro-pro-MMPsMMPs

CytokinesCytokines

Upregulate Upregulate vascular cell vascular cell

MMPsMMPs

Macrophage foam cellsMacrophage foam cells


Macrophage non-MMP proteases?Macrophage non-MMP proteases?• Cathepsins Cathepsins K and S

– have elastolytic activity have elastolytic activity – Expressed by macrophages in atheroma (Sukhova et Expressed by macrophages in atheroma (Sukhova et

al. 1998)al. 1998)• ThrombinThrombin

– generated at sites of disruption, can be generated in generated at sites of disruption, can be generated in the atherosclerotic plaques via tissue factor the 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 networks• Macrophage MMPs may increase the activity of other Macrophage MMPs may increase the activity of other

proteases through: proteases through: – Activation of MMP zymogens - e.g., MMP-3 can activate pro-MMP-1Activation of MMP zymogens - e.g., MMP-3 can activate pro-MMP-1– inactivation of inhibitors - e.g., MMP-1, MMP-3, and MMP-9can inactivation of inhibitors - e.g., MMP-1, MMP-3, and MMP-9can

inactivate alpha 1-antitrypsin, the primary physiologic inhibitor of inactivate alpha 1-antitrypsin, the primary physiologic inhibitor of human leukocyte elastase (Sires et al. 1994human leukocyte elastase (Sires et al. 1994 ); MMP-1, MMP-7, MMP-9, ); MMP-1, MMP-7, MMP-9, and MMP-12 cleave tissue factor pathway inhibitor (Belaaouaj et al, 2000)and MMP-12 cleave tissue factor pathway inhibitor (Belaaouaj et al, 2000)

• Macrophage non-MMPs may increase the activity of Macrophage 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 ?


Ruptured Ruptured plaqueplaqueThe mutual stimulation of generation of active The mutual stimulation of generation of active

MMPs and thrombin may the basis of sustained MMPs and thrombin may the basis of sustained plaque instability, with recurring episodes of plaque instability, with recurring episodes of

plaque disruption and thrombosisplaque disruption and thrombosis

MMPsMMPsThrombinThrombin

MMPsMMPs

ThrombinThrombin

Thrombin Thrombin activates activates latent MMPs latent MMPs (Galis et al., (Galis et al., 1997)1997)

Active MMPs Active MMPs stimulate stimulate generation of generation of thrombin thrombin (Sawicki et (Sawicki et al., 1997)al., 1997)

Proteases and the unstable atherosclerotic plaqueProteases and the unstable atherosclerotic plaque


Are these proteases redundant?Are these proteases redundant?What are the potential actions of all What 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 the MMPs can degrade all the components of the

extracellular matrixextracellular matrix• uPA uPA 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 for Do MMPs provide a common pathway for plaque weakening by other proteases?!?plaque weakening by other proteases?!?


ConclusionsConclusions• Macrophages provide several essential components that Macrophages provide several essential components that

can increase proteolytic activity within atherosclerotic can 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 matrix All circumstantial evidence supports degradation of matrix due to an increased macrophage-related proteolytic activity due to an increased macrophage-related proteolytic activity as a mechanism thorough which macrophage infiltrates as 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!

Health & Medicine

Vp 2002 talk show copy galis