Macrophageinduced proteolysis: how many MMPs and nonMMPs are involved

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Macrophage-induced proteolysis how many MMPs
and non-MMPs are involved??
March 16, 2002 3rd Vulnerable Plaque Symposium
Zorina S. Galis, Ph.D.
Division of Cardiology , Emory University School
of Medicine
Department of Biomedical Engineering
Emory/Georgia Tech, Atlanta GA
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Natural history of human atherosclerosis
Culprit rupture
M. Davies, 1998
Acute cardiovascular events represent a late
stage of arterial remodeling
WEAKENING OF MATRIX SCAFFOLD
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Selected MMP
The matrix metalloproteinase (MMP) family of
enzymes can break-down matrix components
Selected substrates
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First are MMPs expressed in human atheroma?
Lysis of fluorescent substrate
MMP proteins are overexpressed in the vulnerable
shoulders, but are they enzymaticaly active ?
The shoulders of human atherosclerotic plaques
contain active MMPs
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Vulnerable plaques have a high percentage of
macrophage-foam cells
Lumen
Farb Virmani
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Macrophage (MF) foam cells in the shoulders of
human atheroma express MMPs
Immunohistology Detection of MMP-3
Double immunohistology Detection of MMP-1 and MF
(Galis et al., 1994, J Clin Invest)
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Other MMP sightings in the atheroma

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

Macrophage foam cells are associated with
increased MMP expression and activity
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Do MMPs degrade the collagen of the fibrous cap?

• Macrophage MMP-2 and MMP-9 degrade ex vivo the
  collagen of the plaques fibrous cap (Shah et al,
  1995)
• MMP-1 and MMP-13 colocalize in the plaque with
  epitopes expressed by degraded collagen
  (immunohistochemistry, Sukhova et al, 1999)
• MMP-8 colocalizes with epitopes expressed by
  cleaved type I collagen in the shoulders of human
  plaque (immunohistochemistry, Herman et al., 2001)

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Overexpression of interstitial collagenase (MMP-1
) coincides with the places subjected to the
highest tensile stress within the vulnerable
shoulders (Lee et al. 1996)
Bad luck?!?
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Determinants of atherosclerotic plaque stability
Tissue characteristics
Mechanical stress
thin fibrous cap large lipid core (Cheng et al.
1993)
mechanical hot spotscoincide with the weak
points (Lee et al. 1996)
inflammation (Lendon et al. 1991, van der Wal et
al., 1994)
Active degradation of matrix scaffold in the
vulnerable shoulders by MMPs (Galis et al 1994,
Galis et al. 1995)
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Potential modulators of MMP expression and
activity in atheroma

• 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)
• growth factors FGF-2 (Pickering et al 1997)
  VEGF (Wang Keiser 1998)

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Experimental modelfor investigation of
macrophage foam cell MMPs
Subcutaneous granuloma
Balloon angioplasty
Hypercholesterolemic diet
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Macrophage (MF)-derived foam cells (FC) produce
reactive oxygen species (Rajagopalan et al. 1996
JCI)
Macrophage-derived FC activate the zymogen of
MMP-9 in vitro (Galis et al., 1998 Circulation)

Superoxide
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Reactive oxygen species activate latent MMPs
produced by human vascular smooth muscle cells
Xanthine/ Xanthine Oxidase


Pro-MMP-9
Pro-MMP-2
Pro-MMP-2
In vivo?
(Rajagopalan et al. 1996, JCI)
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N-acetyl cysteine (NAC) treatment decreases in
situ MMP-9 expression and activity in
experimental rabbit atheroma
Pro-MMP-9 -
MMP-9 -
MMP-2 -
(Galis et al., 1998 Circulation)
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Lipid lowering therapy may increase plaque
stability by decreasing the oxidative stress
Therapeutic interventions and plaque stability

• improvement of endothelial function
• decreased plaque lipid
• decreased MMP production
• increased plaque stability

• improvement of endothelial function
• decreased MMP production
• increased plaque stability

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Experimental macrophage-rich arterial lesions
(ApoE KO mouse carotid artery ligation)
Macrophage-rich neointima
Normal carotid artery
Atherosclerotic carotid artery
(Lessner et al., unpublished)
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Our studies indicate that arteries with
macrophage-rich lesions undergo enhanced positive
remodeling
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Positively remodeling is associated with plaque
instability
cohort
Remodeling
Schoenhagen et al., 2000 Circulation 101
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(Galis and Khatri, 2002)
Outward arterial remodeling
Normal artery
Foam cell/macrophage-driven
Unstable plaque
Constrictive arterial remodeling
Stable plaque
Smooth muscle cell-driven
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Macrophage-foam cells are key regulators of
MMP-dependent degradation of vascular matrix
Macrophage foam cells
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Macrophage non-MMP proteases?

• Cathepsins K and S
• have elastolytic activity
• Expressed by macrophages in atheroma (Sukhova et
  al. 1998)
• Thrombin
• generated at sites of disruption, can be
  generated in the atherosclerotic plaques via
  tissue factor expressed by macrophage foam cells
  (Wilcox et al. 1989)
• can activate latent MMP-2 (Galis et al., 1997)

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Potential protease networks

• Macrophage MMPs may increase the activity of
  other proteases through
• Activation of MMP zymogens - e.g., MMP-3 can
  activate pro-MMP-1
• inactivation of inhibitors - e.g., MMP-1, MMP-3,
  and MMP-9can inactivate alpha 1-antitrypsin, the
  primary physiologic inhibitor of human leukocyte
  elastase (Sires et al. 1994) MMP-1, MMP-7,
  MMP-9, and MMP-12 cleave tissue factor pathway
  inhibitor (Belaaouaj et al, 2000)

• Macrophage non-MMPs may increase the activity of
  other proteases through
• Activation of MMP zymogens -- e.g., uPA can
  activate pro-MMP-3, thrombin can activate
  pro-MMP-2
• inactivation of inhibitors ?

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Proteases and the unstable atherosclerotic plaque
Active MMPs stimulate generation of thrombin
(Sawicki et al., 1997)
Thrombin activates latent MMPs (Galis et al.,
1997)
Ruptured plaque
The mutual stimulation of generation of active
MMPs and thrombin may the basis of sustained
plaque instability, with recurring episodes of
plaque disruption and thrombosis
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Are these proteases redundant?What are the
potential actions of all these in relation to
plaque rupture?

• MMPs can degrade all the components of the
  extracellular matrix
• uPA activates MMP zymogens (Carmeliet et al.
  1997)
• Thrombin can activate MMP zymogens (Galis et al.
  1997)

Do MMPs provide a common pathway for plaque
weakening by other proteases?!?
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Conclusions

• Macrophages provide several essential components
  that can increase proteolytic activity within
  atherosclerotic plaques
• Produce proteases MMPs, cathepsins, thrombin via
  TF
• Provide activators for proteases ROS, MMPs
• Provide means to inactivate protease inhibitors
  MMPs
• Stimulate production of proteases in vascular
  cells

All circumstantial evidence supports degradation
of matrix due to an increased macrophage-related
proteolytic activity as a mechanism thorough
which macrophage infiltrates precipitate plaque
destabilization, however..
The direct evidence is still missing!