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Molecular mechanisms of Fibrosis:
Targets of Therapy
John P IredaleUniversity of Edinburgh, UK
•The wound healing MFBs of the liver and the hepatic macrophages are key players in progressive and resolving fibrosis.
•The fibrotic liver retains a significant capacity for matrix degradation which is held in check.
•Resolution of fibrosis is characterised by apoptosis of the HSC/MFBsand matrix degradation associated with diminished hepatic TIMP levels.
•Strategies based on harnessing the matrix degrading capacity of the liver and using HSCs and MSCs as delivery vehicles remain attractive.
Take home messages:
Copyright ©2007 American Society for Clinical Investigation
Iredale, J. P. J. Clin. Invest. 2007;117:539-548
Macrophages populate hepatic scars during injury
6wks 8wks 12wks CCl4
Can we demonstrate the influence of macrophages on the fibrotic response in a mechanistic experimental system?
Using the DTr mouse MØ can be effectively andspecifically deleted in progressive CCl4 induced fibrosis
Duffield….Iredale JCI 2005
Fibrosis: Effect of experimentalMacrophage depletion
12 weeks CCl4
+ Macrophages
- Macrophages
Duffield….Iredale JCI 2005
β-galactoside binding lectin
Galectin-3
Found in all 3 cellular compartments
One of a family of 14 currently identified galectins
Galectins highly conserved through evolution
Galectin-3 regulates liver fibrosisGalectin-3-/-WT
Colla
gen
Henderson…Iredale et al, PNAS 2006
Myofibroblast activation is defective in vivoα-
SMA
Galectin-3-/-WT
Henderson…Iredale et al, PNAS 2006
α-SM
Aco
llage
nMacrophage-derived Galectin-3 drives myofibroblast
activation in the kidney following UUO
WT mØs Gal-3-/- mØs
% α
-SMA
WT Gal-3-/-
1234
0
5
*
Proc
ollag
enge
ne ex
pres
sion
WT Gal-3-/-
*
0
2.5
5
7.5%
colla
gen
WT Gal-3-/-0
4
2
6
*
Henderson et al Am J Path 2008
Copyright ©2007 American Society for Clinical Investigation
Iredale, J. P. J. Clin. Invest. 2007;117:539-548
GELATIN SEPHAROSE CHROMATOGRAPHY OFHSC CONDITIONED MEDIA
Gelatinase activity in culturemedia following TIMP-1 removalµg Gelatindegraded/18 hr
46.8
2.4InitialMedia
Post C’graphMedia
% inhibition ofgelatinase activity100
31
Post C’graphMedia + 10%Buffer
Post C’graphMedia + 10%TIMP-1 eluate
Add back of TIMP-1containing fraction
Activated HSC/MFB
TIMP
Matrix degrading metalloproteinase (MMP)
MMP activity inhibiteNo matrix degradatiooccurs
Collagen I-richscar matrix
•HSC and Other NPCs/ICs expressMMPs
•Latent capacity for degradation of fibrillar matrix is held in check
Iredale et al JCI 1992
REVERSIBILITY OF CCl4 MODEL
4 weeks 4 weeks + 10 days recoveryIredale JCI 1998
Collagenase activity during recovery
0
20
40
60
80
100
120
PFO 4d 7d 28d
OHPTIMPCol'nase
TIMP is reducedMatrix degradation occurs
Iredale JCI 1998
21 1 2 7 42 sham0
5
10
15
days afterbile ductligation
days afterbile duct
anastomosis
αSM
A po
siti
ve c
ells
per
high
pow
er fi
eld
Fibrosis Days of recovery
REVERSIBILITY MODEL: NUMBERS OF ACTIVATEDSTELLATE CELLS
Fibrosis
PROGRESSION
HEPATOCYTEKUPFFER CELLINFLAMMATORYCELL
QUIESCENT HSC ACTIVATED HSC APOPTOTIC HSC
RESOLUTION
Number HSCTIMPsCollagenCollagenase
Number HSCTIMPsCollagenCollagenase
Gal 3TGF B1PDGFetc
Products of damaged cells
TIMP
RESPONSE TO LAMIVUDINE IN HEPATITIS B
Wanless 1999
12 Week CCl4 Days of Recovery
0Days 84Days 366Days
Collagen Cross-linking may limit recovery from fibrosis
366Days X-link366Days X-link
0Day X-link
0Days X-link
0Days tTG
Elastin tTG
Issa et al Gastroenterol 2004
Stylised diagrammatic summary of resolution in micronodular cirrhosis
12 Weeks CCl4MICRONODULAR CIRRHOSIS
HV
HV
HVHV
HV
HV
12 Weeks CCl4 + 168d – 366d
MACRONODULAR CIRRHOSIS
PT
X-Linked Elastin rich
Wanless 2000
Evidence for limited matrix degradation in human explant material
Assessing the role of collagen-I in mediating HSC survival
Wild Type collagen I Mutant collagen IMMP MMP
Complete Degradation Persistence
Day 0
Day 28
αSMA S Red αSMA S Red
Issa et al FASEB J 2002
WT rr WT rr WT rr WT rr0.0
0.5
1.0
1.5
PF0 PF4 PF7 PF28
*
*
Mea
n TU
NEL
pos
itive
cel
lsin
the
fibro
tic b
and
/HP
Apoptosis in Cell Populations in WT and r/r mice During Recovery from Fibrosis Determined by TUNEL
Activated HSC
Fibrosis
PROGRESSION RESOLUTION
HEPATOCYTEKUPFFER CELLINFLAMMATORYCELL
QUIESCENT HSC ACTIVATED HSC APOPTOTIC HSC
Number HSCTIMPsCollagenMMPase
Factors favouringsurvival:• Cytokines/soluble
factors• Matrix stabilisation• Cell-cell receptor
stabilisation
Number HSCTIMPsCollagenMMPase
Factors favouringapoptosis:• Death receptor
activation• Withdrawal of
survival factors:• Matrix degradation• Cell receptor
degradation
Fibrosis
PROGRESSION RESOLUTION
KUPFFER CELLMACROPHAGE
QUIESCENT HSC ACTIVATED HSC APOPTOTIC HSC
Number HSCTIMPsCollagenMMPase
Number HSCTIMPsCollagenMMPase
What is the source of the Collagenase and other key MMPs?Is there a role for M’phages/inflamm cells in recovery?
PF0 PF 3660.00
0.01
0.02
0.03
0.04 **
recovery time
cells
/cm
2
C α-SMA Sirius redC α-SMA Sirius red
AA
In the long term, persistent scars are hypocellular:
366d recoverya-SMA in mature scar
Relative paucity of (partic) inflammatory cells
HSC/MFBs express abundant TIMP-1 mRNA, Macrophages express MMPs 12
and 13: in situ
TIMP-1 MMP-12 MMP-13
MMP-13 N 4W 6W 8W 12W
Depletion of scar associated macrophages attenuates resolution
of liver fibrosisPeak fibrosis 7d resolution: control 7d resolution: depletion
Duffield JS et al, J Clin Invest 2005
x 100 x 100 x 100
Effect of conditional macrophage depletion on MMP-13 mRNA: in situ hybridisation
CCl4
CCl4 + depletion
0
5
10
15
20
25
30
35
CCl CCl4 + depletion control
Cells per 10 high power fields
Treatment
CCl4 CCl4 + depletion
*p<0.05
control
Fallowfield et al JI 2007
Fibrosis
PROGRESSION RESOLUTION
HEPATOCYTEKUPFFER CELLINFLAMMATORYCELL
QUIESCENT HSC ACTIVATED HSC APOPTOTIC HSC
Number HSCTIMPsCollagenMMPase
TIMP-1
Proliferative responseto hepatocyte damage
Progressive Fibrosis Resolution of Fibrosis and Parench’Renewal
Hepatocytes
Intact Collagen-I
Activated MFB
MFBSurvivalCollagen-I
MFB apoptosisCollagen-I
Degraded Collagen-I
Proliferative responseto hepatocyte damage
Summary
TIMP/MMP Balance
Mø as regulator and
?Vehicle
Cell RenewalBlock/vehicle
AcknowledgementsN.HendersonT.SethiS.HartlandR.AucottA.PellicoroC.BenyonT.Gordon-WalkerT.KendallJ.FallowfieldR. IssaF. Murphy
S.Forbes
MRC UKWMTBLTCLDFBayerFerring
J.DuffieldS. ClayJ.SavillJ.HughesR.LangS.KraneI. Wanless
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