molecular mechanisms of fibrosis: targets of therapy€¦ · resolution in micronodular cirrhosis...

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