Transcript Effect of HDGF on Hepatic Stellate Cells

義大醫院 醫學研究部
功能型研究室進度報告
Upregulation of Hepatoma-Derived Growth Factor is
Involved in Murine Hepatic Fibrogenesis
實驗室主持人: 高英賢
Liver Fibrosis
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Definition of fibrosis:
as the presence of excessive accumulation of extracellular matrix (ECM) proteins
due to new fiber formation. (WHO expert group, 1978)
Liver fibrosis:
results from chronic damage to liver in conjunction with ECM deposition, which
is a characteristic of most types of chronic liver diseases.
The main causes:
chronic HCV infection,
alcohol abuse,
chronic cholestatic disorders,
non-alcoholic steatohepatitis (NASH)
Fibrous scar formed in fibrotic liver distorts the hepatic architecture, and the
subsequent development of nodules of regenerating hepatocytes defines
cirrhosis, a risk factor for developing hepatocellular cacinoma (HCC).
Changes of collagen composition
from normal to cirrhotic livers
Gressner and Weiskerchen, 2006
Role of Hepatic Stellate Cells (HSCs) in Liver Biology
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HSCs, also named as Ito cells, fat-storing cells, vitamin A-storing cells,
perisinusoidal cells.
Main biological functions: (Sato et al., 2003)
1) Fat and retinoid storage and homeostasis.
2) Remodeling of ECM, by production of both ECM component and
matrix metalloproteinases (MMPs).
3) Production of growth factors and cytokines.
4) Contraction and dilation of sinusoidal lumen in response to
vasoactive substances.
Bataller and Brenner, JCI, 2005
Etiologies of Liver Fibrosis
Chemical
Cholestatic
Ethanol,
hepatotoxins (CCl4,TTA…)
Hepatocyte injury
Bile acid…
Biliary injury
Kupffer cell
activation
Release of free radicals
and signaling cytokines
HSC
activation
Hepatoma-Derived Growth Factor (HDGF)
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HDGF was purified from the conditioned medium of hepatoma-derived cell
line, HuH-7, and has growth-stimulating activity for fibroblasts and
hepatoma cells. (Nakamura et al., 1989)
HDGF has a 36% amino acid homology with the high mobility group 1
(HMG-1) protein involved in DNA-binding and postulated to play a role in
chromosomal replication and transcription. (Nakamura et al., 1994)
It contains a well-conserved N-terminal amino acid sequence, which is
called the HATH (homologous to amino terminus of HDGF) region.
(Nakamura et al., 1994)
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To date, six members were found in HDGF family:
HDGF,
HDGF-related proteins (HRP)-1,
HRP-2 (Izumoto et al., 1997),
HRP-3 (Ikegame et al., 1999),
HRP-4 (Dietz et al., 2002),
Lens epithelial-cell-derived growth factor (LEDGF) (Ge et al., 1998).
Mouse HDGF Protein Family
HATH region
(1~98 a.a.)
(155~170 a.a.)
HDGF
237 a.a. in total
(211~227 a.a.)
HRP-1
283 a.a. in total
(321~363 a.a.)
HRP-2
669 a.a. in total
(136~148 a.a.)
HRP-3
203 a.a. in total
NLS1
NLS2
Bipartite nuclear localizing signal (NLS)
Role of HDGF in Liver Development
(Enomoto et al., 2002)
Role of HDGF in Hepatocellular Carcinoma (HCC)
(Hu et al., 2003)
Involvement of HDGF in Tumorigenesis
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HCC (Hu et al.; 2003 Yoshida et al., 2003 & 2006)
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HDGF is a prognostic marker in non-small-cell lung cancer (Ren et al.,
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2004; Iwasaki et al., 2005).
Involvement in other organ tumors:
colorectum (Lepourcelet et al., 2005),
stomach (Yamamoto et al., 2006),
pancreas (Uyama et al., 2006),
esophagus (Yamamoto et al., 2007), and
gastrointestinal stromal tumors (Chang et al., 2007).
Organogenesis
(Morphogenesis)
Mitogenesis
&
Angiogenesis
Embryogenesis
Tumorigenesis
Since liver fibrosis frequently precedes the
occurrence of HCC …..
Questions ???
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Whether HDGF is involved in the pathogenesis of liver
fibrosis?
Or further, whether HDGF gene knockdown can be an
applicable therapeutic strategy to liver fibrosis?
Specific Aims
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To characterize the expression profile of HDGF during
hepatic fibrogenesis and to clarify its pathological
significance as compared to fibrotic markers.
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To determine the impact of adenovirus-mediated HDGF
gene delivery on liver physiological functions.
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To determine the impact of HDGF overexpression on the
progression of liver fibrosis.
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To elucidate the molecular mechanism by which HDGF
intervenes in hepatic fibrogenesis.
PART I
To characterize the expression profile of
HDGF during hepatic fibrogenesis and to
clarify its pathological significance as
compared to fibrotic markers.
Materials & Methods
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Animals: adult (6~8 w/o) male ICR mice (purchased from National
Lab Animal Center, Taipei), performed under the surveillance of the
Animal Ethics Committee of Kaohsiung Chang Gung Memorial
Hospital.
Biochemical detection for liver function and integrity, such as
aspartate aminotransferase (AST), alanine aminotransferase (ALT),
lactate dehydrogenase (LDH), alkaline phosphotase (ALP), gglutamyltranspeptidase (g-GT), direct bilirubin (D.B.), total bilirubin
(T.B.)
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RT and quantitative real-time PCR (qRT-PCR, Roche)
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Western blotting
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Immunohistochemistry (IHC)
Animal Models for Liver Fibrosis Induction
BDL model
Day 0 (n=6) Day 7 (n=6) Day 14 (n=6)
BDL surgery
CCl4 model
Day 18 (n=6)
Day 0
1 mL/Kg s.c., twice weekly
Day 35 (n=6)
Elevated HDGF gene expression in fibrotic livers
Elevated HDGF protein levels in fibrotic livers
HDGF localization using IHC
Normal livers
BDL, day 7
CCl4, day 18
PART II
To determine the impact of adenovirusmediated HDGF gene delivery on liver
physiological functions.
Animal Treatment
Day 0
Day 14
Ad-GFP (vehicle control)
Day 0
Ad-HDGF
Day 14
Ad-HDGF gene delivery leads to hepatocellular injury and
increases collagen deposition.
Ad-HDGF gene delivery leads to hepatocellular injury and
collagen deposition.
AST levels (U/L)
250
*
*
200
150
100
50
Control Ad-GFP Ad-HDGF
(n=6)
(n=6)
(n=6)
% Area of Collagen Deposition
2D Graph 3
1
*
*
0
Control Ad-GFP Ad-HDGF
(n=6)
(n=6)
(n=6)
Data are expressed as mean ± SEM.
* indicates p<0.05 as compared to control.
PART III
To determine the impact of HDGF
overexpression on the progression of
liver fibrosis.
Animal Treatments
BDL model
Day 0
Day 14
BDL surgery
CCl4 model
Day 35
Day 0
CCl4 s.c. injection (1 mL/Kg) twice weekly
Ad-GFP and Ad-HDGF (109 pfu/mouse) were singly injected through tail
vein 24h before BDL surgery or the first CCl4 injection.
HDGF gene delivery potentiates TGF-b and procollagen a1
protein expression in fibrotic livers.
HDGF gene delivery deteriorates the collagen deposition in
fibrotic livers.
Morphometrical analysis
2D Graph 4
5
4
**
*
3
2
1
0
Control
(n=6)
% Area of Collagen Deposition
% Area of Collagen Deposition
2D Graph 5
d7
d 14
(n=5)
(n=5)
BDL
8
**
***
6
4
2
0
Control
(n=6)
**
*
2
1
0
Control
(n=6)
d 18
d 35
(n=5)
(n=4)
CCl4
% Area of Collagen Deposition
% Area of Collagen Deposition
3
Ad(-)
(n=5)
Ad-GFP Ad-HDGF
(n=5)
(n=5)
BDL
2D Graph 8
2D Graph 6
4
***
5
**
4
***
***
3
2
1
0
Control
(n=6)
Mean ± SEM.
* : p<0.05; ** : p<0.01; ***: p<0.001.
Ad(-) Ad-GFP Ad-HDGF
(n=5)
(n=4)
(n=5)
CCl4
PART IV
To elucidate the molecular mechanism by
which HDGF intervenes in hepatic
fibrogenesis.
Mutual regulation between HDGF and TGF-b in cultured hepatocytes
Schematic summary
Conclusion
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We propose that the unique mutual regulation between HDGF and
TGF-b expression may augment the profibrogenic signaling during
fibrogenesis.
HDGF not only initiates the HSC population expansion at early
stages of liver fibrosis, but also enhances the HSC transformation to
myofibroblasts by up-regulating a-SMA and collagen protein
expression.
Thus, HDGF mechanistically plays an pro-fibrogenic role during
hepatic fibrogenesis.
Thanks for your attention !