inflammation and extracellular proteinases -...
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Inflammation and extracellular proteinases
Questions
• Why focus on metalloproteinases?• What turns them on?• What effect does this have in vitro?• What effect does it have in man?
Thincap
Plaque rupture 75% of MI
Thin fibrous cap (<65 µm), large lipid core, no collagen, macrophages, T-cells Davies, M Circulation 1996; Falk, E JACC 2006;47:C7-12
Foamy macrophages, apoptosis
No collagen
Low collagen
Foam cells
Foam cells make proteasesthat cause plaque vulnerability
van der Wal Cardiovasc Res 1999;41:334-344
Metalloproteinases and TIMPs
TIMPs1-4
24 MMPs
Numbers
MMPs 1,8,13
MMPs (2),9
MMPs 3,7,10,11
MMP 12,19
MMPs 14-7,23,25
MMP class
Interstitial collagenases
Gelatinases (A),B
Stromelysins
Metalloelastase +
Membrane-type MMPs
MMPs cause matrix destruction
Henney, et al. Proc Natl Acad Sci USA, 1991;88:8154-8158.Sukhova et al Circulation 1999;99:2503-9
MMP-1
MMP2378912
1314
EffectMore smc (1) More smc/macrophages (2,3)Fewer smc (2)More macrophages/ less smc (4)More smc/macrophages (5,2)More macrophages, apoptosis, aneurysms (5,2)Less collagen (6)Less collagen (6)
1) ATVB 2006;26:1120 2) ATVB 2001;21:1440 3) PNAS 2005;102:15575 4) Circ Res 2009;105:921 5) Circ 2004;109:1408 6) Circulation 2005;112:2708 7) Circ 2008;117:931
In mice, some MMPs stabilize and others destabilize plaques
Buket Reel Graciela Newby
David Huang
Does inflammation turn MMPs on?A systems approach
Reel, Sala-Newby, Huang and Newby, Br J Pharm, 2011 and unpublished
Inflammation and macrophage diversity
Invasion Matrix degradation
Phagocytosis, KillingCholesterol uptake
LPS, TNFa
IFNg
M1
Exit from lesions Antigen presentation
Fibrosis, Calcification Angiogenesis
Cholesterol release
IL-4IL-13
M2M-CSF
Classicalmonocytes
InflammatoryMonocytes (10%)
CD16- CD16+
Ox-LDL
M0
Foam cells stimulated by LPS + IFNg
Huang, Sala-Newby et. al. unpublished
0
2000
4000
6000
8000
10000
1 2 3
COX2
*
Cop
y nu
mbe
rs /
ngR
NA
M0 M1 M20
10002000300040005000600070008000
1 2 3
MMP-1
*
0
5
10
15
20
1 2 3
MMP-3
*
0
20000
40000
60000
80000
100000
1 2 3
MMP-7
*
0
10
20
30
40
50
1 2 3
MMP-10*
02000400060008000
1000012000
1 2 3
MMP-14*
01020304050607080
1 2 3
MMP-25
*
M0 M1 M2 M0 M1 M2 M0 M1 M2
M0 M1 M2 M0 M1 M2 M0 M1 M20
2000
4000
6000
8000
10000
1 2 3
TIMP-3
*M0 M1 M2
Foam cells stimulated by IL-4
Huang, Sala-Newby et. al. unpublished
0
2
4
6
8
10
12
1 2 3
MMP-11*
050
100150200250300350400450
1 2 3
MMP-12
*
*
Cop
y nu
mbe
rs /
ngR
NA
M0 M1 M2 M0 M1 M2
Effects of classical activators
Huang, Sala-Newby et. al. unpublished
0,1
1
10
100
1000
10000
MMP-1 MMP-10 MMP-12 MMP-14 TIMP-3 COX-2 IKB
LPSIL-1TNFIFN
*
*
**
*
**
*
**
*
***
*
***
*
*
**
Fold
cha
nge
Inflammatory genesMMPs?
Pro-inflammatory Signaling Pathways
Tedgui & Mallat, Physiol Rev 2006
IkBkinase2ERK1/2P38, JNK
PD for ERKSB for p38SP for JNKSC for IKK2LY for PI3K
ABSPI for JAK2
LPS on MAP and IkB kinases
Pretreat DM
SO
PD SB SPLPS
- + + + +
JNK(p)
JNK
p42-44 ERK(p)
P42-44 ERK
p38(p)
p38
LPS - + +
SC-514 - - +
GAPDH
IkBa
NF-kB
I-kB
U UU
P IKK2
NF-kB
Nuclear factor-kB, NF-kB
IL-1, TNF-a,E-selectin,ICAM-1, VCAM-1MCP-1
Effect on MMP-10 protein
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
DMSO PD SB JNKI SC Ly
0
10
20
30
40
50
60
M0M1M2M0M1M2M0M1M2M0M1M2M0M1M2M0M1M2
Rel
ativ
e M
MP-
10 e
xpre
ssio
n
** *
†
MMP-10
GAPDH
Huang, Sala-Newby et. al. unpublished
PD for ERKSB for p38SP for JNKSC for IKK2LY for PI3K
Huang, Sala-Newby et. al. unpublished
0
5
10
15
20
25
30
35
40
M0-
Ut
M1-
Ut
M2-
Ut
M0-
Bga
lM
1-B
gal
M2-
Bga
lM
0-iK
Bv
M1-
iKB
vM
2-iK
Bv
M0-
dnIK
…M
1-dn
IK…
M2-
dnIK
…
Rel
ativ
e M
MP-
10 e
xpre
ssio
n
MMP-10
GAPDH
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
Untransfection β-gal iκB virus dnIKK2 virus
**
Effect of viruses on MMP-10 protein
Effect on MMP-14 protein
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
DMSO PD SB JNKI SC Ly
Huang, Sala-Newby et. al. unpublished
PD for ERKSB for p38SP for JNKSC for IKK2LY for PI3K
0
0,5
1
1,5
2
2,5
3
3,5
4
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
Rel
ativ
e M
MP
-14
exp
ress
ion
*
**
†
MMP-14
GAPDH
IFNg Signaling Pathway
Tedgui & Mallat, Physiol Rev 2006
ABSPI for JAK2
Inflammatory genesMMPs?
MMPs stimulated by IFNg
Huang, Sala-Newby et. al. unpublished
-20
0
20
40
60
80
100
120
140
MMP-9 MMP-12 MMP-14 SOCS-3
DMSO DMSO + 20micoM IFN IFN + 20micoM
Perc
enta
ge o
f IFN
gal
one
* *
*
**
* * *
Effect of JAK-2 inhibitor, ABSPI
M1
M3
M7
M8
M10
M11
M12M14
Incr
easi
ng m
RN
A ex
pres
sion
M17
M25
IKK2
↑LPS (TNFa, IL-1) + INFγ ↑IL4
JNK and/or ERK
T2T1 T3
↔
M23
The human macrophage MMP map
Br. J. Pharm. 2011 and unpublished
Jak-2
Which MMPs are turned on in foam cells in vivo?
Foam cell macrophages and non-foamy macrophages from cholesterol or chow-fed rabbits
NFM
FCM
Chase et al. ATVB 2002;22:765-771
0
5
10
15
20
25
30
MMP 1
MMP 3
MMP 12
MMP 14
TIMP 3
Prot
ein
leve
ls(a
rbitr
ary
units
)
FCM NFM
Several MMPs are increased and TIMP 3 decreased in foam cells
*
*
*
**
Chase et al. ATVB 2002;22:765-771
TIMP-3- TIMP-3+
Rabbit foam cells are a mixture of TIMP3+ and TIMP3-
4 week rabbit plaqueRAM11 red, TIMP-3 brown
Zaltsmann et al 1999, unpublished and Johnson ATVB 2008; 28:1647
Jason JohnsonBHF, IRF
MCP-1
TIMP-3 stops invasion
T3Top
T3Bottom
MCP-1
Bottom
MMP14
MMP14
Johnson ATVB 2008; 28:1647
+ TIMP3Top
Matrigel layer
Anti-MMP-14 also stops invasion
MMP14+TIMP-macrophages invade
Chemoattractant
MatrigelLayer
Migrated foam cells
MMP-14 + TIMP-3 –
Johnson et al ATVB 2008; 28:1647
Gelatinolytic activity
0
20
40
60
80
100
Control rTIMP-3 MT1-MMP Bab
% c
ells
exp
ress
ing
gela
tinol
ytic
act
ivity
**
Johnson ATVB 2008; 28:1647
Apoptosis (and proliferation) also greater in MMP-14+, TIMP-3-
0
50
100
150
Control rTIMP-3 MT1-MMP Bab
Ap
op
tos
is (
%
co
ntr
ol)
*
Johnson ATVB 2008; 28:1647
TIMP-3
Dying cells
*
Invasive destructive apoptotic foam cells - cap thinning and core expansion
Thincap
No collagen
Does it translate to mice?
• Studies on mouse monocyte macrophages in vitro
• Studies in ApoE null mice
Subcutaneous spongesBracheocephalic arteries
Jason JohnsonBHF, IRF
Karina di Gregoli
Monocyte invasion into s.c sponges and ApoE null plaques
0100200300
Control MT1-MMP Blocking Ab
**
Mac
roph
ages
S.C sponges
0
2
4
6
8
Control MMP14 BAb
Mac
roph
ages
/ sec
tion
**
BCA plaques
Bristol Coronary Biobank
Jenkins, Johnson, Pasterkamp, Newby, unpublished
CD68
MMP-14
CD68
MMP-14
Unstable Stable
Steve White Nick Jenkins
I II III IV VI V VII VIII
** *
0
20
4
0
60
80
100
Role of TLR2
Monaco C. Circulation. 2009;120(24):2462-2469.
Role of TLR2
Monaco C. Circulation. 2009;120(24):2462-2469.
Subsequent death of patient
<45%TIMP-3
>45%MMP-14
Collaboration with Vincent Scholtes and Gerard Pasterkamp
Conclusions• Some metalloproteinases cause instability
others not likely• Turned on by inflammation via LPS,
TNF, IL-1, CD40L • More selectively by IFNg and IL-4• More invasive and destructive more likely
to undergo apoptosis• Consitent with effects observed in vivo
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