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Understanding Root Cause: 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)
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J Clin Invest. 115: 209, 2005
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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”
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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
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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
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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.
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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
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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.
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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
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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
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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
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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
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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
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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