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10/1/12 1 Understanding Root Cause: Pathogenesis of Hepatic Fibrosis Hepatitis C Virus Inflammation Fibrosis Cirrhosis Mild inflammation

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Page 1: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

10/1/12

1

Understanding Root Cause: Pathogenesis of Hepatic Fibrosis

Hepatitis C Virus

Inflammation

Fibrosis Cirrhosis

Mild inflammation

Page 2: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Non-alcoholic Fatty Liver Disease

Steatosis Steatohepatitis

Fibrosis Cirrhosis

Fibrotic Liver Diseases

Patients Mice

HCV, HBV (Post necrotic cirrhosis)

Secondary biliary fibrosis

Alcoholic liver disease

Autoimmune hepatitis

Non-alcoholic steatohepatitis (NASH)

Carbon tetrachloride (CCl4) Thioacetamide (TAA) Mouse with humanized liver with hepatitis infection Bile duct ligation

Intragastric ethanol

Heterologous serum

Diet induced obesity Choline deficient diet (CDAA)

Page 3: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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J Clin Invest. 115: 209, 2005

Page 4: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Hepatic Stellate Cell (HSC) activation in culture

ACTIVATION

activated HSC

quiescent HSC

Vit-A autofluorescence a-sma

ECM

Modifiers of hepatic fibrosis using knock out mice

Susceptibility to fibrosis

IL-6, IL-10

iNOS

Plasminogen

Interferon g Adiponectin

Telomerase

Bcl-xL

SOCS1 (+/-)

Keratin

Knock out:

TNFR1, TLR4, CD14

Angiotensin R1a

NADPH oxidase

TGFb1, Smad3, MMP13

p21

FGF1/2, FGFR4

Leptin, OB-R

Dopamine bhydroxylase

FasL, Cathepsin B

C5 Hepatology 37: 653, 2003

Increased Decreased

“Fibrogenic” “Protective”

Page 5: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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NAD(P)H-oxidase

Oxidative stress induces liver fibrosis

NAD(P)H-oxidase

P450IIE1

Ethanol/acetaldehyde AngII, Bile salts

HCV proteins

ROS

Activation Proliferation

Activation Apoptosis

Fibrosis

Examples of kos – NADPH oxidase, angiotensin R1a

Renin Angiotensin Pathway in HSCs Induces ROS and Hepatic Fibrosis

Hepatic fibrosis

Angiotensinogen

Angiotensin-I

Angiotensin-II

AT1 AT2

Renin

ACE

ARB

ACE-inh

NADPH oxidase

O2 O2 . p67phox p47phox

p

gp91phox

p22phox

Ca+2 flux HSC activation ECM production

Page 6: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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NOXs in hepatic stellate cells

NOX1

• Non-phagocytic form of NOX • Expressed in VSMCs and fibroblasts • Induced by Ang II, Leptin, PDGF

NOX2

• Classic phagocytic form of NOX • Expressed in macrophages • Inducible

NOX4 • Regulated at level of transcription • Increased in inflammation and fibrosis • Induced by TGFb

Liver fibrosis is blocked by NOX 1/4 inhibitor GKT137831

Siri

us r

ed p

ositi

ve a

rea

(%)

0

5

10

15

TIM

P-1

mR

NA

(fol

d)

0

50

100

colla

gen

1(I)

mR

NA

(fol

d)

0

40

80

120

C

E

WT

SOD1mu CCl4

CCl4

CCl4

CCl4

Vehicle

NOX1/4 inhibitor

D

CCl4

WT SOD1mu

Vehicle

oil oil CCl4

-SMA

-actin

oil CCl4 oil CCl4

WT SOD1mu

NOX1/4 inhibitor

TGF-

mR

NA

(fol

d)

0

5

10

15

* *

*

WT SOD1mu

CCl4

CCl4

CCl4

CCl4

Vehicle

NOX1/4 inhibitor

A B

CCl4

- - + + - - + + NI

oil

WT

SOD1mu

CCl4

- - + + - - + + NI

oil CCl4

- - + + - - + + NI

oil CCl4

- - + + - - + + NI

oil

* *

*

* *

*

* *

*

WT

SOD1mu

Page 7: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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TGF1 induces liver fibrogenesis

§  Activates HSCs

§  Stimulates synthesis of ECM

• Collagens

• Glycoproteins

• Proteoglycans

§  Inhibits ECM degradation • Inhibits MMPs

• Stimulates protease inhibitors

§  Increases TGF1 mRNA

Examples of ko mice – TGFb1, Smad3, MMP13,TLR4

Integrin αvβ6 activates TGFβ

RDG TGFb

LTBP1

b6

RDG

av

TGFbR

Sheppard D. Integrin-mediated activation of latent transforming growth factor beta. Cancer Metastasis Rev. 2005 Sep;24(3):395-402.

Page 8: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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HSC

Hepatocyte

Activated HSC

Phagocytosis

MIP-2 KC TGFb1

Apoptotic bodies

Apoptosis

Hepatocyte

Kupffer cell

F I B R O S I S

Hepatocyte apoptosis induces hepatic fibrosis

Examples of ko mice – Fas, Cathepsin B, Bcl-xL, IKKb

TLR4-mutant mice display strongly reduced fibrosis after BDL TLR4WT TLR4mutant

Sham Sham

BDL BDL

TLR4WT TLR4mutant

Siri

us r

ed s

tain

ing

S

MA

-sta

inin

g

BDL BDL

Sham Sham

TLR4WT

0

100

200

300

400

sham BDL

sham BDL 0

5

10

15

20

Hyd

roxy

prol

ine

(mg/

g liv

er)

Siri

us r

ed p

os-a

rea

(%)

TLR4mutant

Sirius red

Hydroxyproline

Page 9: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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LPS

LPS activates TLR4 to induce fibrosis

Kupffer cell quiescent HSC

TLR4 Bambi

Chemokines

Adhesion molecules

TGF TGFb-RI

HSC activation

Liver fibrosis

Collagen deposition

Studies of modifier genes in patients

Modifier genes: Genes that alters the expression of other gene(s) and affects phenotype. Genetic polymorphisms: Stable variations in DNA that are observed in 1% or more of the population (i.e. SNPs). Association studies: epidemiological studies assessing the role of genetic polymorphisms in disease susceptibility and/or progression.

Page 10: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Modifiers of hepatic fibrosis in patients

Hepatology 37:653, 2003

HCV – HFE, Angiotensinogen, TGFb1, CTLA-4, MnSOD, TNFa, IL10, MTP, MCPI/Z, MMP 1/3/9, RANYES, Kertin 8/18, a AT, C5, DDX5, CPT-1A, IFN8

ALD – HFE, TGFb1, CTLA-4, MnSOD, TNFa, IL10, CD14, GSTMI/T1

NASH – HFE, Angiotensinogen, TGFb1, MnSOD, TNFa, MTP

blue – genes that modify multiple liver diseases

Problems: Inadequate sample size, different populations, statistical stringency, small genetic effects

Celera Whole Genome Scan for Cirrhosis in HCV

Gastro, 2006 Hepatology, 2007

Page 11: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Predictors

Gene (Chr)

Risk of genotype or phenotype

Frequency (Caucasian

Patients)

SNP1b

SNP2

SNP3

SNP4

SNP5 SNP6

SNF7

Sex

Age

Alcohol

AZIN1 (Chr 8)

TLR4 (Chr 9)

TRPM5 (Chr 11)

None (Chr 15)

None (Chr 1) None (Chr 3)

AQP2 (Chr 12)

GG

CC

CT, CC

AG, AA

GG AG, AA

GG

Male

Older

≥ 50 g/day

86.9%

87.3%

63.6%

41.6%

72.3% 18.5%

66.7%

69.3%

N/A

32.9%

Modifier Genes of Hepatic Fibrosis in HCV (Celera, Hepatol. 2008)

Chronic Inflammation leads to fibrosis

Activated

HSC activation

WBC recruitment

Kupffer cell activation

TNF- IL-1 MIP-1

LPS TLR4

Myofibroblast

INJURY FIBROSIS

TGF-b Ang II Leptin ROS

Ko mice –TNF-, TLR4, CD14, LBP

Page 12: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Reversal of CC14-induced Hepatic Fibrosis

Iredale, JCI 102:538, 1998

CC14

Post 7d

Collagen Activated HSCs

Fibrosis and Resolution

Fibrosis Resolution

ECMs

Collagenase ___

TIMPs

Activated HSCs

Page 13: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Removing known stimuli of fibrogenesis reverses fibrosis in patients

•  Inflammation – autoimmune hepatitis

•  Ethanol – alcoholic liver disease

•  Copper – Wilson’s disease

•  Iron – hemochromatosis

•  Schistosomes – schistosomiasis

•  Biliary Obstruction – 2ndary biliary cirrhosis

•  Hepatitis B and C – viral hepatitis

•  NASH – obesity, diabetes

Lamivudine decreases activated HSCs

Pre-LAM Post-LAM

J Hepatol 35:749,2001

a smooth muscle actin staining

Page 14: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Rates of Regression of HCV cirrhosis with SVR

D’Ambrosio et al. Hepatology. Volume 56, pages 532, 2012

Gastric Bypass Surgery— Decreased hepatic fibrogenesis in

NAFLD

•  Decreased steatosis

•  Decreased activated hepatic stellate cells

•  Decreased mRNAs for intermediate biomarkers of fibrosis: collagen α1(I), TGFβ1, TIMP-1, and αSMA

Pre-GBS

Post-GBS

Page 15: Understanding Root Cause: Pathogenesis of Hepatic Fibrosis · 2012-10-01 · Pathogenesis of Hepatic Fibrosis Hepatitis C Virus! Inflammation! Fibrosis! Cirrhosis! Mild inflammation!

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Fibroblast Quiescent HSC

Fibrocyte

Proliferation

Fibrogenesis

Chronic Liver Injury

Angiotensin PDGF Leptin

Apoptosis

SMA+ Myofibroblast

TGFb1

ROS

NADPH oxidase

Pathogenesis of Hepatic Fibrosis