Upregulation of Hepatoma-Derived Growth Factor is Upregulation of Hepatoma-Derived Growth Factor is Involved in Murine Hepatic FibrogenesisInvolved in Murine Hepatic Fibrogenesis

實驗室主持人實驗室主持人 :: 高英賢高英賢

義大醫院 醫學研究部義大醫院 醫學研究部功能型研究室進度報告功能型研究室進度報告

Liver FibrosisLiver Fibrosis

Definition of fibrosis: Definition of fibrosis:

as the presence of excessive accumulation of extracellular matrix as the presence of excessive accumulation of extracellular matrix (ECM) proteins due to new fiber formation. (ECM) proteins due to new fiber formation. (WHO expert group, 1978)(WHO expert group, 1978)

Liver fibrosis:Liver fibrosis:

results from chronic damage to liver in conjunction with ECM results from chronic damage to liver in conjunction with ECM deposition, which is a characteristic of most types of chronic liver deposition, which is a characteristic of most types of chronic liver diseases.diseases.

The main causes:The main causes:

chronic HCV infection,chronic HCV infection,

alcohol abuse,alcohol abuse,

chronic cholestatic disorders,chronic cholestatic disorders,

non-alcoholic steatohepatitis (NASH)non-alcoholic steatohepatitis (NASH)

Fibrous scar formed in fibrotic liver distorts the hepatic architecture, Fibrous scar formed in fibrotic liver distorts the hepatic architecture, and the subsequent development of nodules of regenerating and the subsequent development of nodules of regenerating hepatocytes defines cirrhosis, a risk factor for developing hepatocellular hepatocytes defines cirrhosis, a risk factor for developing hepatocellular cacinoma (HCC).cacinoma (HCC).

Changes of collagen composition Changes of collagen composition from normal to cirrhotic liversfrom normal to cirrhotic livers

Gressner and Weiskerchen, 2006

Role of Hepatic Stellate Cells (HSCs) in Liver BiologyRole of Hepatic Stellate Cells (HSCs) in Liver Biology

HSCs, also named as HSCs, also named as ItoIto cells, fat-storing cells, vitamin A-storing cells, fat-storing cells, vitamin A-storing cells, perisinusoidal cells.cells, perisinusoidal cells.

Main biological functions: Main biological functions: (Sato et al., 2003) 1) Fat and retinoid storage and homeostasis.1) Fat and retinoid storage and homeostasis. 2) Remodeling of ECM, by production of both ECM component 2) Remodeling of ECM, by production of both ECM component

andand matrix metalloproteinases (MMPs).matrix metalloproteinases (MMPs). 3) Production of growth factors and cytokines.3) Production of growth factors and cytokines. 4) Contraction and dilation of sinusoidal lumen in response to4) Contraction and dilation of sinusoidal lumen in response to vasoactive substances.vasoactive substances.

Bataller and Brenner, JCI, 2005

Etiologies of Liver FibrosisEtiologies of Liver Fibrosis

Hepatocyte injuryHepatocyte injury

ChemicalChemicalEthanol, Ethanol,

hepatotoxins (CClhepatotoxins (CCl44,TTA,TTA……))

Biol

ogical

Biol

ogical

HCV, H

BV, p

aras

ites

HCV, H

BV, p

aras

ites……

CholestaticCholestaticBile acidBile acid……

Metabolic

Metabolic

Fatty acid, glucose,

Fatty acid, glucose,……Biliary injuryBiliary injury

Kupffer cellKupffer cellactivationactivation

Release of free radicals Release of free radicals and signaling cytokinesand signaling cytokines

HSCHSCactivationactivation

Hepatoma-Derived Growth Factor (HDGF)Hepatoma-Derived Growth Factor (HDGF)

HDGF was purified from the conditioned medium of hepatoma-HDGF was purified from the conditioned medium of hepatoma-derived cell line, HuH-7, and has growth-stimulating activity for derived cell line, HuH-7, and has growth-stimulating activity for fibroblasts and hepatoma cells. fibroblasts and hepatoma cells. (Nakamura et al., 1989)

HDGF has a 36% amino acid homology with the high mobility HDGF has a 36% amino acid homology with the high mobility group 1 (HMG-1) protein involved in DNA-binding and postulated group 1 (HMG-1) protein involved in DNA-binding and postulated to play a role in chromosomal replication and transcription. to play a role in chromosomal replication and transcription. (Nakamura et al., 1994)

It contains a well-conserved N-terminal amino acid sequence, It contains a well-conserved N-terminal amino acid sequence, which is called the which is called the HATHHATH ((hhomologous to omologous to aamino mino tterminus of erminus of HHDGF) region. DGF) region. (Nakamura et al., 1994)

To date, six members were found in To date, six members were found in HDGF familyHDGF family:: HDGF,HDGF, HDGF-related proteins (HRP)-1,HDGF-related proteins (HRP)-1, HRP-2 HRP-2 (Izumoto et al., 1997), HRP-3 HRP-3 (Ikegame et al., 1999), HRP-4 HRP-4 (Dietz et al., 2002), Lens epithelial-cell-derived growth factor (LEDGF) Lens epithelial-cell-derived growth factor (LEDGF) (Ge et al., 1998).

Mouse HDGF Protein Family

HDGFHDGF

HRP-1HRP-1

HRP-2HRP-2

(1~98 a.a.)

NLS1NLS1

(155~170 a.a.)

(211~227 a.a.)

(321~363 a.a.)

237 a.a. in total

283 a.a. in total

669 a.a. in total

HRP-3HRP-3(136~148 a.a.)

203 a.a. in total

Bipartite nuclear localizing signal (NLS)Bipartite nuclear localizing signal (NLS)

NLS2NLS2

HATH regionHATH region

Role of HDGF in Liver DevelopmentRole of HDGF in Liver Development

(Enomoto et al., 2002)

Role of HDGF in Hepatocellular Carcinoma (HCC)Role of HDGF in Hepatocellular Carcinoma (HCC)

(Hu et al., 2003)

Involvement of HDGF in TumorigenesisInvolvement of HDGF in Tumorigenesis

HCC HCC (Hu et al.; 2003 Yoshida et al., 2003 & 2006)

HDGF is a prognostic marker in non-small-cell lung cancer HDGF is a prognostic marker in non-small-cell lung cancer (Ren et al., 2004; Iwasaki et al., 2005).

Involvement in other organ tumors:Involvement in other organ tumors: colorectum colorectum (Lepourcelet et al., 2005),, stomach stomach (Yamamoto et al., 2006),, pancreas pancreas (Uyama et al., 2006),, esophagus esophagus (Yamamoto et al., 2007), and , and gastrointestinal stromal tumors gastrointestinal stromal tumors (Chang et al., 2007)..

MitogenesisMitogenesis&&

AngiogenesisAngiogenesis

OrganogenesisOrganogenesis(Morphogenesis)(Morphogenesis)

EmbryogenesisEmbryogenesis TumorigenesisTumorigenesis

Questions ???Questions ???

Whether HDGF is involved in the pathogenesis of Whether HDGF is involved in the pathogenesis of liver fibrosis?liver fibrosis?

Or further, whether HDGF gene knockdown can be Or further, whether HDGF gene knockdown can be an applicable therapeutic strategy to liver fibrosis?an applicable therapeutic strategy to liver fibrosis?

Since liver fibrosis frequently precedes the occurrence of HCC …..

Specific AimsSpecific Aims

To characterize the expression profile of HDGF during To characterize the expression profile of HDGF during

hepatic fibrogenesis and to clarify its pathological hepatic fibrogenesis and to clarify its pathological

significance as compared to fibrotic markers.significance as compared to fibrotic markers.

To determine the impact of adenovirus-mediated To determine the impact of adenovirus-mediated

HDGF gene delivery on liver physiological functions.HDGF gene delivery on liver physiological functions.

To determine the impact of HDGF overexpression on To determine the impact of HDGF overexpression on

the progression of liver fibrosis.the progression of liver fibrosis.

To elucidate the molecular mechanism by which To elucidate the molecular mechanism by which

HDGF intervenes in hepatic fibrogenesis.HDGF intervenes in hepatic fibrogenesis.

PART IPART I

To characterize the expression profile To characterize the expression profile of HDGF during hepatic fibrogenesis of HDGF during hepatic fibrogenesis

and to clarify its pathological and to clarify its pathological significance as compared to fibrotic significance as compared to fibrotic

markers.markers.

Materials & MethodsMaterials & Methods

Animals: adult (6~8 w/o) male ICR miceAnimals: adult (6~8 w/o) male ICR mice (purchased from (purchased from National Lab Animal Center, Taipei), performed under the National Lab Animal Center, Taipei), performed under the surveillance of the Animal Ethics Committee of Kaohsiung surveillance of the Animal Ethics Committee of Kaohsiung Chang Gung Memorial Hospital.Chang Gung Memorial Hospital.

Biochemical detection for liver function and integrity, such Biochemical detection for liver function and integrity, such as aspartate aminotransferase (as aspartate aminotransferase (ASTAST), alanine ), alanine aminotransferase (aminotransferase (ALTALT), lactate dehydrogenase (), lactate dehydrogenase (LDHLDH), ), alkaline phosphotase (alkaline phosphotase (ALPALP), ), -glutamyltranspeptidase (-glutamyltranspeptidase (-GT-GT), ), direct bilirubin (direct bilirubin (D.B.D.B.), total bilirubin (), total bilirubin (T.B.T.B.))

RT and quantitative real-time PCR (qRT-PCR, Roche)RT and quantitative real-time PCR (qRT-PCR, Roche)

Western blottingWestern blotting

Immunohistochemistry (IHC)Immunohistochemistry (IHC)

BDL modelBDL model

CClCCl44 model model

Day 18 (n=6) Day 35 (n=6)Day 0

1 mL/Kg s.c., twice weekly1 mL/Kg s.c., twice weekly

Day 7 (n=6) Day 14 (n=6)Day 0 (n=6)

BDL surgeryBDL surgery

Animal Models for Liver Fibrosis InductionAnimal Models for Liver Fibrosis Induction

Elevated HDGF gene expression in fibrotic livers Elevated HDGF gene expression in fibrotic livers

Elevated HDGF protein levels in fibrotic livers Elevated HDGF protein levels in fibrotic livers

HDGF localization using IHCHDGF localization using IHC

Normal liversNormal livers BDL, day 7BDL, day 7 CClCCl44, day 18, day 18

PART IIPART II

To determine the impact of To determine the impact of adenovirus-mediated HDGF gene adenovirus-mediated HDGF gene

delivery on liver physiological delivery on liver physiological functions.functions.

Animal TreatmentAnimal Treatment

Day 14Day 0

Day 14Day 0

Ad-GFP (vehicle control)

Ad-HDGF

AdAd-HDGF-HDGF gene delivery leads to hepatocellular gene delivery leads to hepatocellular injury and increases collagen deposition.injury and increases collagen deposition.

AdAd-HDGF-HDGF gene delivery leads to hepatocellular gene delivery leads to hepatocellular injury and collagen deposition. injury and collagen deposition.

Data are expressed as mean ±Data are expressed as mean ± SEM.SEM.** indicates p<0.05 as compared to indicates p<0.05 as compared to control.control.

PART IIIPART III

To determine the impact of HDGF To determine the impact of HDGF overexpression on the progression overexpression on the progression

of liver fibrosis.of liver fibrosis.

Animal TreatmentsAnimal Treatments

BDL modelBDL model

CClCCl44 model model

Day 14Day 0

BDL surgery

Day 35Day 0

CCl4 s.c. injection (1 mL/Kg) twice weekly

Ad-GFP and Ad-HDGF (10Ad-GFP and Ad-HDGF (1099 pfu/mouse) were singly injected pfu/mouse) were singly injected through tail vein 24h before BDL surgery or the first CClthrough tail vein 24h before BDL surgery or the first CCl44 injection.injection.

HDGFHDGF gene delivery potentiates TGF- gene delivery potentiates TGF- and and procollagen a1 protein expression in fibrotic livers. procollagen a1 protein expression in fibrotic livers.

HDGFHDGF gene delivery deteriorates the collagen gene delivery deteriorates the collagen deposition in fibrotic livers. deposition in fibrotic livers.

Morphometrical analysisMorphometrical analysis

Mean ±Mean ± SEM.SEM.** : p<0.05; : p<0.05; **** : p<0.01; : p<0.01; *** ***: p<0.001.: p<0.001.

PART IVPART IV

To elucidate the molecular mechanism To elucidate the molecular mechanism by which HDGF intervenes in hepatic by which HDGF intervenes in hepatic

fibrogenesis.fibrogenesis.

Mutual regulation between HDGF and TGF-Mutual regulation between HDGF and TGF- in cultured in cultured hepatocyteshepatocytes

Schematic summarySchematic summary

ConclusionConclusion

We propose that the unique mutual regulation between HDGF and TGF-We propose that the unique mutual regulation between HDGF and TGF- expression may augment the profibrogenic signaling during fibrogenesis.expression may augment the profibrogenic signaling during fibrogenesis.

HDGF not only initiates the HSC population expansion at early stages of HDGF not only initiates the HSC population expansion at early stages of liver fibrosis, but also enhances the HSC transformation to myofibroblasts liver fibrosis, but also enhances the HSC transformation to myofibroblasts by up-regulating by up-regulating -SMA and collagen protein expression.-SMA and collagen protein expression.

Thus, HDGF mechanistically plays an pro-fibrogenic role during hepatic Thus, HDGF mechanistically plays an pro-fibrogenic role during hepatic fibrogenesis. fibrogenesis.

Thanks for your attention !Thanks for your attention !