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Reel, Sala-Newby, Huang and Newby, Br J Pharm, 2011 and unpublished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 unpublished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 unpublished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