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國立中山大學 生物科學系

博士論文

探討肝癌衍生生長因子在肝臟纖維化機制中所扮演的病理性角色

Investigation on the Pathological Role of Hepatoma-Derived

Growth Factor in Hepatic Fibrogenesis

研 究 生：高英賢 撰

指導教授：戴明泓 博士

趙大衛 博士

中華民國 九十八年八月

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Contents

Abbreviations ……………………………… 2

Abstract in Chinese ……………………………… 4

Abstract in English ……………………………… 6

Introduction ……………………………… 8

Materials and Methods ……………………………… 25

Results ……………………………… 43

Discussion ……………………………… 55

Future Perspective ……………………………… 63

References ……………………………… 65

Tables ……………………………… 80

Figures ……………………………… 86

Appendixes (Author’s CV and publications) ….…… 117

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Abbreviations

ALT Alanine aminotransferase

ALP Alkaline phosphotase

AST Aspartate aminotransferase

α-SMA α-Smooth muscle actin

BDL Bile duct ligation

BrdU 5-Bromo-2’-deoxyuridine

CCl4 Carbon tetrachloride

ECM Extracellular matrix

ELISA Enzyme-linked immunosorbent assay

ET-1 Endothelin-1

ERK Extracellular signal-regulated kinase

FAK Focal adhesion kinase

FBS Fetal bovine serum

γ-GT γ-Glutamyltranspeptidase

HCC Hepatocellular carcinoma

HCV Hepatitis C virus

HDGF Hepatoma-derived growth factor

HMG High mobility group

HRP HDGF-related protein

HSCs Hepatic stellate cells

HSP47 Heat shock protein 47

ILK Integrin-linked kinase

LDH Lactate dehydrogenase

MAPK Mitogen-activated protein kinase

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MMP Matrix metalloproteinase

mRNA Message Ribonucleic Acid

NO Nitric oxide

PDGF Platelet-derived growth factor

pfu Plaque forming unit

PI3K Phosphatidyl-3-inositol kinase

PPAR Peroxisome proliferator-activator receptor

qRT-PCR Quantitative reverse transcription and polymerase chain

reaction

TGF-β Transforming growth factor-β

TIMP Tissue inhibitor of metalloproteinase

TNF-α Tumor necrosis factor-α

VEGF Vascular endothelial growth factor

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中文中文中文中文摘要摘要摘要摘要

肝臟纖維化的病理病變常導致器官功能障礙而致命，臨床上是肝

膽腸胃道疾病中被探討的主要課題之一。目前認為肝臟纖維化的過程

是組織受到各種不同的慢性刺激所誘發的一種傷口癒合反應。纖維化

組織具有過量細胞外基質蛋白的堆積，這些堆積的蛋白常會破壞肝臟

的正常結構，最後導致器官病理性破壤。肝癌衍生生長因子

(hepatoma-derived growth factor)是一純化自肝癌細胞株培養上清

液之生長因子，與陸續發現的另五個相關蛋白組成一組蛋白家族。肝

癌衍生生長因子在胚胎發育時期的肝組織中大量表現，被認為在細胞

分裂、器官生成、胚胎形成、與腫瘤發生等過程中扮演多功能角色。

先前的研究中已證實肝癌衍生生長因子在肝癌組織中有過度表現的現

象，其表現程度與肝癌細胞的生長呈正相關，並可作為一良好預後指

標性蛋白。由於肝臟纖維化常在肝癌臨床病徵發現之前即已出現，因

此本研究主要探討利用膽管結紮手術與注射四氯化碳化學物質所誘發

之肝臟纖維化之兩種小鼠動物模式中，肝癌衍生生長因子在肝臟纖維

化過程中可能扮演之病理性角色。研究結果發現，肝癌衍生生長因子

在這兩種動物模式中的表現量均與纖維化過程呈時間依賴性變化，並

且在纖維化肝臟組織主要表現於血管周邊的肝實質細胞中。此外，其

表現量亦與肝內乙型轉形生長因子(Transforming growth factor;

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TGF-β)與第一型前膠原蛋白(pro-collagen type I)的增加呈一致性。在

誘發肝臟纖維化前若先行送入攜帶肝癌衍生生長因子基因之腺病毒載

體，使其大量表現，則會促使 TGF-β表現增加與 pro-collagen type I分

子過度沉積。在體外作用模式中，發現當肝細胞培養在具有膠原蛋白

披覆之培養盤時，可觀察到肝癌衍生生長因子與 TGF-β間正向地相互

調節彼此之分子表現量。此結果顯示肝癌衍生生長因子與 TGF-β間所

產生之促纖維化訊息傳遞具膠原蛋白依存性（collagen- dependent）。

其很可能在肝臟纖維化初期，即藉由此種惡性循環模式作用而加速肝

臟的纖維化病變。再者，以肝癌衍生生長因子重組蛋白作用於培養肝

細胞，可顯著地刺激培養肝臟星狀細胞中之 BrdU uptake與α-smooth

muscle actin、pro-collagen type I分子表現量增加，此意味著肝細胞生

成之肝癌衍生生長因子可能藉由 paracrine的作用模式，活化肝臟星狀

細胞。本研究證實肝癌衍生生長因子在肝臟纖維化過程中扮演著促纖

維化的角色，預期在未來利用阻斷肝癌衍生生長因子的作用路徑可發

展為預防或治療慢性肝臟疾病之有效策略之一。

關鍵辭關鍵辭關鍵辭關鍵辭：肝臟纖維化、肝癌衍生生長因子、乙型轉形生長因子、肝細

胞。

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Abstract

Liver fibrosis, a major medical problem with significant morbidity and

mortality, is considered as a wound-healing response to a variety of chronic

stimuli. It is characterized by an excessive deposition of extracellular

matrix (ECM) proteins, which disrupts the normal architecture of liver and

ultimately leads to pathophysiological damage to liver. Hepatoma-derived

growth factor (HDGF), a growth factor originally purified from hepatoma

cells, is highly expressed in fetal hepatocytes and hepatoma. It is known to

play multifunctional roles in mitogenesis, organogenesis, embryogenesis,

and tumorigenesis. Its expression correlates with the proliferating state of

hepatocellular carcinoma (HCC) and serves as a prognostic factor. Since

liver fibrosis frequently occurs prior to HCC development, the specific aim

of this study is to investigate the role of HDGF in the progression of liver

fibrosis by using animal models of mice receiving either bile duct ligation

surgery or carbon tetrachloride administration. Quantitative real-time PCR

and Western blotting analysis showed a significant elevation of HDGF

expression in both models. HDGF levels correlated with progression of

liver fibrosis in a time-dependent manner as well as paralleled with the

expression of other two fibrotic markers, transforming growth factor-β1

(TGF-β1) and pro-collagen type I, in fibrotic livers. Intriguingly, the

over-expressed HDGF protein was localized mainly in perivenous

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hepatocytes of fibrotic livers. Besides, adenovirus-mediated HDGF gene

delivery potentiated the production of TGF-β1 and pro-collagen type I,

thereby enhancing the intrahepatic collagen matrix deposits as evidenced

by Sirius red stain and morphometrical analysis. In cultured hepatocytes,

TGF-β1 and HDGF mutually up-regulated their de novo synthesis only

when grown on collagen-coated matrix, strongly suggesting that the

TGF-β1- and/or HDGF-driven pro-fibrogenic signaling is

collagen-dependent and a vicious circle may exist at the initial stage of

hepatic fibrogenesis. Moreover, administration with recombinant HDGF

stimulated BrdU uptake and synthesis of both α-smooth muscle actin and

pro-collagen type I in cultured hepatic stellate cells, implicating that a

mode of paracrinal action lies between these two cell types. In conclusion,

HDGF plays a pro-fibrogenic role during liver fibrosis and blockade of

HDGF pathway may potentially constitute the preventive or therapeutic

strategies for chronic liver diseases.

Keywords: Liver fibrosis; Hepatoma-derived growth factor; Transforming

growth factor-β; Hepatocytes.

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Introduction

Liver fibrosis is a major medical problem with significant morbidity and

mortality. Liver cirrhosis, as an end-stage consequence of fibrosis, is

characterized by nodule formation and altered hepatic function. Clinical signs of

liver cirrhosis vary widely, from asymptomatic to complete liver failure. Once

complications begin to develop, which may include ascites, esophageal varices

or jaundice, progressive deterioration of liver function cannot be avoided

(Tsukada et al., 2006). Particularly in Taiwan, hepatitis caused by viral infections,

including hepatitis B and hepatitis C, represent the primary cause of liver fibrosis,

which differs from the epidemiological statistics in the United States, where

chronic ethanol consumption is the leading cause (Chedid et al., 2004; Siegmund

and Brenner, 2005; Siegmund et al., 2005).

Stimuli for hepatic fibrosis include autoimmune disorders, drug-induced

hepatotoxicity, helminthic infection, iron or copper overload, and biliary

obstruction. All these etiologies eventually result in cirrhosis and many

complications, such as portal hypertension, liver failure, and hepatocellular

carcinoma (HCC). There is increasing evidence that non-alcoholic steatohepatitis

is a leading cause of fibrosis. If left untreated, fibrosis can progress to cirrhosis,

ultimately leading to liver failure and possible death.

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Fibrosis can be considered as a wound-healing response to a variety of

chronic stimuli (Bissell, 1998; Tsukada et al., 2006), and characterized by an

excessive deposition of extracellular matrix (ECM) proteins which include three

major families of proteins: glycoproteins, collagens, and proteoglycans. The

most commonly associated characteristic of fibrosis is increased deposition of

collagen, specifically the fibrillar collagens. Although the number of different

collagens identified in the liver continues to increase, the two most commonly

associated collagens that have been demonstrated and discussed to the greatest

extent are types I and III collagens (Friedman, 1997; Tsukada et al., 2006).

During liver fibrosis, altered collagen synthesis at both mRNA and protein levels

was observed, with a dramatic increase in type I collagen along with smaller, but

significant increase in type III collagen. At advanced stages, liver contains

approximately 6 times more ECM than normal livers. The excess deposition of

ECM molecules disrupts the normal architecture of liver by dense bands of

collagens that link vascular structures and surround islands of regenerating

parenchymal cells, and ultimately leading to pathophysiologically functional

damage to the organ (Bataller and Brenner, 2005). These changes are

characteristics of cirrhosis. Advanced fibrosis and cirrhosis are generally

considered to be irreversible conditions even after removal of the injurious agent.

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However, the irreversibility of liver fibrosis still remains debatable to date.

Role of Hepatic Stellate Cells in Liver Fibrosis

Hepatic stellate cells (HSCs), formally named Ito cells, fat-storing cells, or

lipocytes, are the primary cell type responsible for production of collagen-like

ECM molecules (Gressner, 1996). In this regard, activation of HSCs has long

been identified as one of the main features of liver fibrosis. In normal liver,

HSCs remain in a quiescent state and constitutively synthesize low levels of

matrix proteins. Following liver injury, HSCs undergo a complex transformation

or activation process. Overall, three major events occurring in HSCs potently

promote the fibrogenic response of the cells:

Firstly, HSCs change morphologically from a quiescent, vitamin A-storing

cell to an activated, myofibroblast-like cell (Eng and Friedman, 2000; Bataller

and Brenner, 2005; Tsukada et al., 2006). The activated HSCs are characterized

by a loss in the cellular droplets and vitamin A stores, appearance of cytoskeletal

protein α-smooth muscle actin (α-SMA), and an increase of the appearance of

endoplasmic reticulum (Mak et al., 1984; Mak and Lieber, 1988).

Secondly, HSCs change their pattern of gene expression (Mann and Smart,

2002), thereby resulting in a dramatic increase in the synthesis and deposition of

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ECM molecules, mainly type I and III collagens. In addition, a dramatic increase

in expression of heat shock protein 47, a collagen-specific molecular chaperone,

is also induced in HSCs during collagen biosynthesis, which is believed to be

closely associated with the excess ECM deposition in fibrotic livers (Masuda et

al., 1994; Brown et al., 2005). It has been known that the intrahepatic

accumulation of ECM proteins is attributable not only to increased ECM

synthesis but also to decreased ECM degradation. Alterations in activity of

ECM-degrading matrix metalloproteinase (MMP) enzymes, to date, are now

known to be closely associated with the tissue remodeling during the progression

of liver fibrosis (Huang et al., 2005; Kornek et al., 2006), while the major cause

is mainly due to the overexpression of their specific inhibitors, tissue inhibitors

of metalloproteinase (TIMPs) (Arthur and Iredale, 1994; Torres et al., 1999;

Leroy et al., 2004; Nie et al., 2006). Accordingly, these cells become directly

fibrogenic.

Thirdly, proliferation rate of HSCs increases following cell activation,

which effectively amplifies the fibrogenic cell population. The cell DNA

synthesis and cellular proliferation increase following activation (Gabele et al.,

2005). Therefore, the cellular parameters mentioned above are frequently used as

indicators for fibrotic severity of livers in both in vivo and in vitro surveys.

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Meanwhile, effective treatments aiming at inhibiting either fibrogenic or

proliferative activities of activated HSCs have been previously proposed

(Albanis and Friedman, 2006; Greupink et al., 2006; Liu et al., 2006; Prosser et

al., 2006).

In addition to the direct contribution of activated HSCs to the development

and progression of liver fibrosis, HSC activation is associated with an increase in

cell contractility, which leads to increased portal blood pressure (portal

hypertension) by either constricting individual sinusoid or contracting the

cirrhotic livers as a whole (Racine-Samson et al., 1997; Rockey, 2001). Among

the factors affecting tissue contractility, endothelin-1 (ET-1) is the major

stimulant for HSCs (Racine-Samson et al., 1997). Activated HSCs produce nitric

oxide, a physiological antagonist of ET-1 (Rockey and Chung, 1995). The net

contractile activity of HSCs in vivo reflects the relative balance of these

counteracting factors. The imbalanced shift from balanced status to ET-1

hyperresponsiveness increases sinusoidal resistance and contraction when liver

disease aggravates (Gupta et al., 1998; Rockey et al., 1998). Similar to other

features of activated HSCs, increasingly complex levels of regulatory

mechanisms that modulate HSC contractility have been revealed, including the

activation of ET-1 by a converting enzyme (Friedman, 1993). All those findings

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suggest ET-1 signaling mediators as promising new therapeutic targets.

Hepatic cell type other than HSCs may also have fibrogenic potential

(Ramm et al., 1998). A complex interplay among different cell types takes place

during hepatic fibrogenesis. Hepatocytes are considered the targets for various

hepatotoxic agents, including hepatic viruses, bacterial toxins, alcohol

metabolites, and bile acids (Bataller and Brenner, 2005). Injured hepatocytes

may release reactive oxygen species and fibrogenic mediators, thereby inducing

recruitment of inflammatory cells into liver. The apoptosis of hepatocytes

subsequently activates the fibrogenic activities of liver myofibroblasts (Canbay

et al., 2004). Moreover, the pro-fibrogenic factors released from infiltrated

lymphocytes (Casini et al., 1997) or Kupffer’s cells (Maher, 2001), the

intrahepatic macrophages, may activate HSCs to synthesize and secrete

collagens.

Cytokines Involved in Liver Fibrosis

Cytokines regulating the inflammatory response to liver injury have been

previously demonstrated to modulate hepatic fibrogenesis both in vivo and in

vitro (Bonniaud et al., 2005; Luedde and Trautwein, 2008). Conversely, activated

HSCs also secrete inflammatory cytokines to activate infiltrating inflammatory

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cells in a mode of vicious circle (Maher, 2001; Aleffi et al., 2005). Among the

well-known pro-fibrogenic cytokines, transforming growth factor-β (TGF-β) is

proven as a key regulator in hepatic fibrogenesis both clinically and

experimentally. TGF-β is a pleiotropic cytokine that is ubiquitously expressed by

all cells and tissues in body. Through its effects on cell growth, differentiation,

motility, adhesion, and apoptosis, TGF-β is an influential regulator in

determining cell fate and in tissue morphogenesis (Bursch et al., 1993; Cain and

Freathy, 2001; Herrera et al., 2004).

TGF-β plays a pivotal role in the fibroproliferative changes following tissue

damage in many vital organs and tissues, including liver, lung, kidney, skin, heart,

and arterial wall (Border and Noble, 1994; Massague and Chen, 2000; Leask and

Abraham, 2004). High levels of TGF-β are often found in sera of patients with

liver fibrosis and it has been implicated as a mediator of fibrosis in many liver

diseases (Bataller and Brenner, 2005; Tsukada et al., 2006). In the progress of

hepatic fibrosis, TGF-β is produced by a number of nonparenchymal cells, such

as activated HSCs, Kupffer’s cells, and sinusoidal endothelial cells (Bataller and

Brenner, 2005). Additionally, it is believed that release of TGF-β by necrotic

hepatocytes may be one of the first group of signals to activate adjacent

quiescent HSCs. When treated with TGF-β1, HSCs transform to myofibroblasts

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and produce more abundant ECM proteins (Garcia-Trevijano et al., 1999), while

ECM degradation is suppressed (Yang et al., 2003).

Since TGF-β plays central roles in both inflammatory and fibrotic

pathophysiological states in livers, TGF-β signaling has been designated as

anti-inflammatory and potentially anti-fibrogenic targets. Thus, strategies aimed

at disrupting the synthesis and bioavailability of TGF-β1 or further at interfering

its signaling notably attenuate fibrosis in animal model (Breitkopf et al., 2005;

Liu et al., 2006) as well as in several human fibrotic diseases (Border and Noble,

1994; Tsukada et al., 2006). Indeed, these findings have major translational

implications for therapeutic strategies aiming at not only preventing fibrosis, but

also preserving organ function.

Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs

and is up-regulated in fibrotic livers (Pinzani et al., 1989). Both

dominant-negative soluble PDGF-β receptor (Borkham-Kamphorst et al., 2004a)

and anti-sense against PDGF-B chain (Borkham-Kamphorst et al., 2004b) inhibit

HSC activation and experimentally attenuate liver fibrosis.

Tumor necrosis factor-α (TNF-α), an important pro-inflammatory mediator,

is frequently linked to the pathogenesis of liver fibrosis. The gene expression of

both TNF-α and monocyte chemoattractant protein-1, which are involved in

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Kupffer cell activation and migration, decreases in the liver of TNF receptor

(p55)-knockout mice (Kitamura et al., 2002), suggesting an indirect role in

regulating HSC activation.

Peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway has

been demonstrated to regulate HSC activation and experimental liver fibrosis

(Miyahara et al., 2000). PPAR-γ ligands inhibit the fibrogenic actions in HSCs

and attenuate liver fibrosis in vivo (Marra et al., 2000), while TNF-α inhibits the

PPARγ transactivity in cultured HSCs by diminishing the DNA binding activity

of PPARγ to PPARγ responsive element and ERK1/2-mediated phosphorylation

of Ser82 of PPARγ (Sung et al., 2004).

All above-mentioned cytokines may directly or indirectly modulate

pro-fibrogenic signaling and have been considered as potential targets for

anti-fibrotic strategies. (Friedman, 2004; Breitkopf et al., 2005; Gressner and

Weiskirchen, 2006; Prosser et al., 2006)

Hepatoma Derived Growth Factor (HDGF)

HDGF was first purified from the conditioned medium of human

hepatoma-derived cell line, HuH-7, by Nakamura’s group in 1989. It possesses

growth-stimulating activity for fibroblasts and hepatoma cells (Nakamura et al.,

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1989). The HDGF protein stimulates not only the growth of HuH-7 cells in

chemically defined serum-free medium, but also the DNA synthesis of Swiss

mouse 3T3 fibroblasts, suggesting that it may be an autocrine growth factor. Not

surprisingly, this putative growth factor was later found to play an important role

in the autonomous growth of hepatoma and may lead to useful diagnostic or

therapeutic approaches to human hepatoma (Nakamura et al., 1989; Hu et al.,

2003).

Researches focusing on HDGF progress quite slowly. Gene cloning for

human HDGF from the cDNA library of HuH-7 cells was done until 1994, on the

basis of its N-terminal amino acid sequence (Nakamura et al., 1994). The amino

acid sequence deduced from this 2.4 kilo-base pair cDNA revealed that it

contains 240 amino acids and lacks a signal peptide sequence for secretion

(Nakamura et al., 1994; Izumoto et al., 1997). In addition, HDGF cDNA

sequence was found to have a basic motif, homologous to the reported consensus

sequences for bipartite nuclear localization signal, indicating that HDGF may

function as a nuclear protein. As expected, the subcellular locations of HDGF

protein were later demonstrated in both cytoplasm and nucleus, and the action of

nuclear translocation is critical to its mitogenicity (Everett et al., 2001; Enomoto

et al., 2002a; Everett and Bushweller, 2003).

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The evidence from Nakamura’s group that HDGF shares high homology

(23.4% amino acids identity in its carboxyl terminal acidic region) with high

mobility group (HMG)-1 protein suggests that HDGF may have similar

functional roles to HMG. Proteins (Izumoto et al., 1997). Thus, HDGF was

previously proposed to be a member of HMG family due to sequence

homologies, heparin binding capacity, the size of molecular mass, and its

ubiquitous expression pattern. HMG chromosomal proteins are non-histone

proteins found in nuclei of higher eukaryotes (Bustin et al., 1990) and may play a

role in chromosomal replication (Kuehl et al., 1985) and transcriptional

regulation (Thanos and Maniatis, 1992). The HMG-1 proteins are present in both

cytoplasm and nucleus by the subcellular fractionation and the

immunofluorescent studies (Einck and Bustin, 1985). In confluent or slowly

growing cells, most of HMG-1 protein is found in cytoplasm, whereas, in

actively dividing cells, found in the nucleus (Mosevitsky et al., 1989). Similarly,

the subcellular locations of HDGF protein may be closely associated with its

mitogenic activities and other unknown functions (Nakamura et al., 1989;

Nakamura et al., 1994).

Role of HDGF in Tumorigenesis

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The earlier studies showing that HDGF is highly and ubiquitously expressed

in fetal developing organs have suggested that HDGF is involved in tissue

morphogenesis and organogenesis (Oliver and Al-Awqati, 1998; Everett, 2001;

Everett et al., 2001; Enomoto et al., 2002a; Enomoto et al., 2002b). The bipartite

nuclear localizing signals in HDGF is functionally associated with its nuclear

translocation, through which it exhibits mitogenic (Everett et al., 2004),

motogenic (Kishima et al., 2002), and pro-angiogenic activities (Okuda et al.,

2003; Everett et al., 2004; Zhang et al., 2006a). The involvement of HDGF in

tumorigenesis was discovered in early 2000s. Matsuyama et al. noticed a

correlation between HDGF gene expression and reduced radiosensitivity in

esophageal cancer and claimed that HDGF could be a novel marker for

predicting effectiveness of radiotherapy in clinical cases (Matsuyama et al.,

2001). Later on, an increasing number of research works focused on

investigating the role of HDGF in tumorigenesis. Evidence emerges that HDGF

is involved in tumorigenesis within various organs, such as non-small-cell lung

cancer (Zhang et al., 2006a) and gastric carcinoma (Yamamoto et al., 2006),

claiming that HDGF can be a powerful prognostic marker. Not surprisingly, the

role of HDGF in stimulating hepatoma cell growth became clear because

anti-sense oligonucleotides to HDGF suppress hepatoma cell proliferation in

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vitro (Kishima et al., 2002). In the meantime, Tai’s group presented in vivo

evidence revealing a close association between HDGF expression and the

occurrence of HCC (Hu et al., 2003), consistent with the reports of Nakamura’s

group (Yoshida et al., 2003; Yoshida et al., 2006).

In addition to its direct stimulation on tumor cell growth, the mechanism of

HDGF-induced tumor formation in vivo also encompasses induction of VEGF

and its direct effect on angiogenic activity (Okuda et al., 2003; Zhang et al.,

2006a), indicating its stimulatory role in metastasis. The involvement of HDGF

in non-small-cell lung cancer (Ren et al., 2004; Iwasaki et al., 2005) and gastric

carcinoma (Yamamoto et al., 2006) has also been delineated and its expression

level was found to be prognostically associated with tumor malignancy. More

recently, the evidence that HDGF-siRNA-treated tumors exhibited markedly

reduced blood vessel formation and increased necrosis suggests that HDGF is

involved in anchorage-independent growth, cell invasion, and formation of

neovasculatures in non-small-cell lung cancer (Uyama et al., 2006), implicating

multifunctional roles of HDGF in the process of tumor development. Taken all

those previous findings together, it is strongly suggested that HDGF could be

used as a target gene for anti-cancer therapy.

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Rationale and Specific Aims for This Study

Current knowledge indicates that certain patients who are chronic carriers of

hepatitis C virus (HCV) will progress to chronic active hepatitis with cirrhosis,

HCC and end-stage liver disease (Imperial, 1999). The overall incidence is

increasing (about 20% of those chronically infected with HCV develop cirrhosis

over 20 years) due to this chronic viral disease (Chitturi and George, 2000;

Benvegnu and Alberti, 2001), while the severity of portal fibrosis has been

known to correlate with the development of HCV-related carcinogenesis (Ikeda

et al., 1998). Researchers have been seeking for useful markers of liver fibrosis

to monitor patients at risk of progressive fibrosis (Wong et al., 1998) and it is a

reasonable concept that genes involved in ECM turnover and immune response

are implicated in the transition from mild to moderate fibrosis (Asselah et al.,

2005)

Although recent evidence has confirmed that HDGF overexpression is seen

in fibrotic lungs and it positively regulates lung epithelial cell proliferation,

(Mori et al., 2004) the role of HDGF in hepatic fibrogenesis is not completely

defined as yet. In this regard, hypoxia-induced VEGF upregulation is commonly

found in early stages of fibrosis and is essential for the development of liver

fibrosis. (Rosmorduc et al., 1999; Corpechot et al., 2002; Yoshiji et al., 2003)

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Given that liver fibrosis frequently occurs prior to HCC development (Giannelli

et al., 2003) and that HDGF, found to be involved in steatohepatitic induction

before tumor formation in mice (Yoshida et al., 2003) and correlate with the

proliferating states of HCC (Hu et al., 2003; Yoshida et al., 2003; Yoshida et al.,

2006), represents a novel prognostic factor for patients with HCC who had

undergone surgery, we hypothesized that HDGF may participate in the process

of liver fibrosis, and set forth to investigate its role in liver fibrogenesis.

For the purpose of investigating the mechanisms of hepatic fibrogenesis,

rodents have been widely used for experimental models of inducing hepatic

fibrosis in studies involving chronic liver diseases (Tsukamoto et al., 1990). Thus,

a diversity of rodent models have been applied for this experimental purpose and

can be categorized into (1) induction by hepatotoxins and hepatocarcinogens

such as carbon tetrachloride (CCl4), dimethylnitrosamine, thioacetamide, and

furan; (2) the hepatotoxin plus nutrient, alcohol; (3) a high fat-low choline-low

protein diet; (4) immunologic agents such as heterologous serum or bacterial cell

wall products; and (5) obstructive jaundice and biliary cirrhosis by common bile

duct ligation (BDL) (Wasser and Tan, 1999; Bataller and Brenner, 2005).

Therefore, in this study we particularly chose two animal models for examining

the pathological role of HDGF in hepatic fibrogenesis that is characterized by

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prominent inflammation and hepatocyte necrosis. The first model, BDL, is

recommended as a useful method capable of reproducibly inducing progressive

biliary fibrosis in mice with prominent hepatocyte necrosis and severe

inflammation. The second one, treatment with CCl4, is also known to have an

advantage to induce fibrosis in rats in a relatively short period (less than 4 weeks)

with slight inflammation and hepatocyte necrosis (Ala-Kokko et al., 1992;

Wasser and Tan, 1999).

Therefore the specific aims of this study were as follows:

I. To characterize the expression profiles of HDGF during hepatic fibrogenesis

and to clarify its pathological role and significance in comparison to fibrotic

markers.

II. To determine the impact of adenovirus-mediated HDGF gene delivery on

liver physiological functions.

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

liver fibrosis, by scrutinizing whether adenovirally mediated HDGF gene

delivery could potentiate the progression of experimentally BDL- or CCl4-

induced liver fibrosis.

After confirming the role played by HDGF, we continued to undergo in vitro

experiments:

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IV. To elucidate the possible molecular mechanism by which HDGF intervenes

in hepatic fibrogenesis.

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Materials and Methods

Animals and Experimental Design

Adult and healthy 6-8 week-old ICR male mice and littermate controls, purchased

from the National Laboratory Animal Center in Taipei, were housed kept at 20–22 ◦C

under 12 h light-dark cycle with free access to water and food. Animal care and all

experimental interventions were performed in accordance with Laboratory Animal

Welfare Act, and under the surveillance of the Animal Ethics Committee of Kaohsiung

Chang Gung Memorial Hospital.

The flow chart for animal experiments in this study is shown in Figure 1.

Experimental mice (N=66) were randomly divided into eleven groups.

Part I. Five groups of mice were used to examine the expression profiles of HDGF and

fibrotic markers:

(1) Normal control group (n=6);

(2) Mice received BDL surgery alone were harvested at day 7 (n=6);

(3) Mice received BDL surgery alone were harvested at day 14 (n=6);

(4) Mice with CCl4 administration alone were harvested at day18 (n=6);

(5) Mice with CCl4 administration alone were harvested at day35 (n=6).

Part II. Two groups of mice were used to evaluate the biological and pathological

effects of adenoviral administration:

(6) Mice were administered with green fluorescent protein (GFP) gene-carried

adenovirus (Ad-GFP) alone for vehicle control and harvested at day 14 (n=6);

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(7) Mice were administered with adenovirus-vehicled HDGF gene (Ad-HDGF) alone

and harvested at day 14 (n=6).

Part III. Four groups of mice were used to determine the effects of adenoviral HDGF

gene delivery on the progression of liver fibrosis:

(8) Mice were administered with Ad-GFP one day before BDL surgery and harvested at

day 14 (n=6);

(9) Mice were administered with Ad-HDGF one day before BDL surgery and harvested

at day 14 (n=6);

(10) Mice were administered with Ad-GFP one day before initiation of CCl4 injection

and harvested at day 35 (n=6);

(11) Mice were administered with Ad-HDGF one day before initiation of CCl4 injection

and harvested at day 35 (n=6).

Liver Fibrosis Induction

Induction of liver fibrosis was implemented by two methods, using BDL surgery

or chemically subcutaneous administration of CCl4, respectively.

For BDL surgery, the surgical procedure was performed under sterile conditions.

After anesthesia with intramuscular injection of Rompun® (an anesthetic and a muscle

relaxant for animals; Bayer, Leverkusen, Germany) (50 mg/Kg), and Ketalar (ketamine

hydrochloride; Parke-Davis, Taipei, Taiwan) (50 mg/Kg), abdominal skin was shaved

and sterilized with an iodine solution. Each mouse underwent laparotomy. A 10 mm

incision was made along upper abdominal midline. Midline laparotomy was performed

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for exploring the hepatic hilum and identifying extrahepatic bile duct. Under the

dissecting microscope, common bile duct was then isolated, doubly ligated and

transected between two ligatures. The incision was closed with two layers of interrupted

absorbable silk sutures. After operation, mice were kept in individual cage until

sacrifice.

For CCl4 injection, mice were subcutaneously administered with a dose of 1

mL/Kg of mixture with mineral oil (1:1) twice weekly for 5 consecutive weeks.

Sample Collection

Animals were sacrificed at 7 and 14 days after BDL and the parallel

adenovirus-treated mice were sacrificed at 14 days to collect serum and liver tissues.

Alternatively, blood and tissue samples were collected at 18 and 35 days after initiation

of CCl4 injection. All procedures were carried out in accordance with institutional

guidelines.

In brief, the mice were sacrificed under overdosed anesthesia followed by a

laparotomy. The blood samples (about 3 ml) were collected by cardiac puncture using a

23 gauge needle attached to a 5-ml syringe, and allowed for clotting at room

temperature before centrifugation for 10 min at 2,500 g×. The sera after separation were

aliquoted and stored at −80 ◦C until further measurements of biochemical analysis and

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ELISA detection of TGF-β1.

Liver tissues were harvested and either frozen rapidly on liquid nitrogen for

storage at −80 °C or fixed with PBS-buffered 10% formalin solution followed by

paraffin embedding until further staining processes for regular histopathology or

immunohistochemistry. The fresh tissues for isolation of both RNA and protein were

stored at −80 ◦C.

Biochemical Analysis

Serum specimens were tested for the indicators of inflammation and hepatic

function integrity, including aspartate aminotransferase (AST), alanine

aminotransferase (ALT), total bilirubin (TB), direct bilirubin (DB), alkaline

phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltranspeptidase (γ-GT)

by the automatic analyzer in the Division of Clinical Biochemical Laboratory,

Kaohsiung Chang-Gung Memorial Hospital.

Cloning, Expression, and Purification of Recombinant Human HDGF Protein

Generation and purification of the recombinant 6×-histidine-tagged HDGF protein

and anti-HDGF were carried out as previously described. (Hu et al., 2003). In brief,

human HDGF cDNA was cloned from a human fetal brain cDNA library (Stratagene,

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La Jolla, CA) by polymerase chain reaction (PCR). The PCR primers used to clone the

human HDGF cDNA were designed based on the HDGF sequence in the GenBank

database (accession number, NM_004494) as forward primer, 5’-GCCATGTCGCG

ATCCAACCGGCAGAA-3’ and reverse primer, 5’-CTACAGGCTCTCATGA

TCTCTG-3’). After DNA sequencing analysis, the PCR-amplified HDGF cDNA was

subcloned into the NdeI and XhoI sites of the pET15b vector (Novagen, Madison, WI)

and transformed into BL-21 cells (DE3, pLysS; Novagen) and the transformed cells

were grown at 37ºC until log phase (OD600nm~0.5-0.9). Subsequently, 1 mM IPTG was

added into culture to induce protein expression and continue to grow for another 3 hr at

30ºC. The cell pellet was harvested by centrifugation at 5000 rpm for 10 min at 4ºC,

resuspended in NETN buffer (20 mM Tris pH 8.0, 0.5% NP-40, 100 mM NaCl, 1mM

EDTA) containing protease inhibitors, and then homogenized by sonication. After

centrifugation at 12000 rpm for 20 min at 4 ºC, the supernatants were collected and

incubated with NTA-agarose affinity column (Qiagen, Hilden, Germany) to absorb

His-tagged proteins, respectively, at 4ºC for 30 min. The recombinant proteins absorbed

onto the beads were eluted with NETN containing 20 mM glutathione (reduced form;

Sigma) or 10 mM imidazole, and subsequently desalted on a G25 Sephadex column

(Amersham Pharmacia, Little Chalfont, United Kingdom). The recombinant protein

was passed through Detoxi-Gel (Pierce Biotechnology, Rockford, IL) to minimize

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contamination by endotoxin. The potency of recombinant HDGF protein has been

previously determined by its in vitro stimulatory effects on proliferation and migration

of NIH-3T3 fibroblast as shown in figure 2.

Generation and Purification of Replication-Deficient Adenoviruses

The recombinant replication-deficient adenoviruses containing green fluorescent

protein (Ad-GFP) and HDGF genes (Ad-HDGF) were generated and purified as

previously described (Tai et al., 2003a). In brief, the full length human HDGF and

antisense HDGF cDNA were subcloned into adenovirus transfer vector, pCA13

(Microbix Inc, Canada). The pCA13-HDGF and pCA13-antisense HDGF vector were

cotransfected into 293 cells with pJM17, a plasmid containing the entire type 5

adenovirus genome with E1-insertion and E3 deletion, by calcium phosphate protocol

to generate recombinant virus through homologous recombination (Graham and Prevec,

1995). The virus plaques were picked and verified by checking cytopathic effect, PCR,

and western blot prior to amplification. The virus was amplified in 293 cells, purified

by two rounds of cesium chloride gradient ultracentrifugation, and dialyzed against

buffer containing 10 mM Tris, pH 7.5, 1 mM MgCl2, 10% glycerol at 4 ℃. The titer of

virus solution was determined by measuring optical density at wavelength of 260 nm

and plaque forming assay in 293 cells before storage at -80 ℃.

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For adenoviral infection experiments, the Ad-GFP or Ad-HDGF was diluted in

saline immediately before use and administered through tail veins at doses of 1×109

plaque-forming units (pfu) each mouse, under anesthesia with ether at 24 h prior to the

BDL surgery or the first CCl4 injection.

RNA Isolation and Quantitative Reverse Transcription and Polymerase Chain

Reaction (qRT-PCR)

Total RNA was extracted from liver tissues and cultured cells using a

commercially available Trizol solution (Invitrogen, Gaithersburg, MD) , according to

the manufacturer’s instructions. Afterwards, it was dissolved in diethylpyrocarbonate

(DEPC)-treated water and was quantified by a spectrophotometer.

Two micrograms of total RNA was used for reverse transcription reaction with

Superscriptase II (Invitrogen) using oligo-dT and random primers. Complementary

DNA (cDNA) was synthesized from total RNA in a range of about 100ng in 20µl of

reaction mixture using a first strand cDNA kit. The reaction mixture consisted of

1×PCR buffer, 5mM MgCl2, 1mM dNTP, 50U RNase inhibitor, 20U AMV reverse

transcriptase, and 1 µg of both oligo(dT)15 and random hexamer as primers (Invitrogen,

San Francisco, USA). The mixture was incubated at 25℃ and then at 42°C for 60 min,

95°C for 5 min, and 4°C for 5 min. Then gene-specific primers were used for

amplification of mRNA expression of interested genes. PCR products were loaded on a

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1.8 % agarose gel for electrophoresis, stained with ethidium bromide and further

visualized by UV illumination. The visualized gels were recorded and analyzed on a

digital imaging system (Alpha Imager 2000, Alpha Innotech Corp., San Leandro, CA).

PCR yields for β-actin after 25-cycle amplification were used for checking the qualities

of RNA isolation and RT reaction efficiency and for internal control.

To quantify the gene expression levels of β-actin, HDGF, α-SMA, collagen α1(I),

and TGF-β in liver extracts, one twentieth of cDNA generated was used as template for

real time PCR analysis. Amplification and detection was done by a LightCycler DNA

Master SYBR Green I kit (Roche Applied Science, Mannheim, Germany) in the

LightCycler Detection System (Roche Applied Science). PCR reaction was carried out:

one cycle of 95°C for 10 min, 45 cycles of 95°C for 15 s, 60°C for 5 s, and 72°C for 20

s. After amplification, a final melting curve protocol was performed to determine the

specificity of PCR reaction. The primer sequences (and GenBank accession nos.) were

listed as follows:

For β-actin gene (NM_01101),

5’-TCCTGTGGCATCCACGAAACT-3’ (forward)

5’-GAAGCATTTGCGGTGGACGAT-3’ (reverse);

For HDGF gene (NM_004494),

5'-CCGGATTGATGAGATGCCTGA-3’ (forward)

5’-TTGTTGGGCTTGCCAAACT-3’ (reverse);

For α-SMA gene (NM_009841),

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5’-TGTGCTGGACTCTGGAGATG-3’ (forward)

5’-GATCACCTGCCCATCAGG-3’ (reverse);

For TGF-β1 gene (NM_021578),

5’-CGTCAGACATTCGGGAAGC-3’ (forward)

3’-CAGCCACTCAGGCGTATCA-3’ (reverse);

For collagen α1 (I) gene (NM_019178),

5’-ACGTCCTGGTGAAGTTGGTC-3’ (forward)

5’-ACCAGGGAAGCCTCTCTCTC-3’ (reverse).

Protein Extraction and Western Blotting Analysis

Total protein extracts from homogenized rat liver tissues and from cultured cells

were lysed in ice-cold RIPA buffer (consisting of 50 mM Tris-HCl, pH 7.4, 5 mM

EDTA, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl) in the presence of

protease inhibitor cocktail (Roche, Indianapolis, IN). After centrifugation, the protein

concentrations in supernatants were determined by the Coomassie protein assay kit

(Pierce Biotechnology, Rockford, IL).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

Western blotting were performed as previously described (Hu et al., 2003). In brief, an

equal amount (50 µg) of total protein for each lane was subjected to SDS-PAGE, using

8% or 12% acrylamide gels under reducing conditions at 120 V for 2 h. Proteins were

subsequently electrotransferred onto a polyvinylidene difluoride (PVDF) membrane

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(Millipore, Bedford, MA, USA) following conventional protocols. Blots were blocked

in 5% skimmed milk in PBS-T (PBS, pH 7.4, with 0.1% Tween-20) for 1h at room

temperature, followed by incubation with primary antibodies diluted in PBS-T at room

temperature or at 4℃ overnight. The detecting antibodies were those raised against

HDGF (1:1,000 dilution, rabbit polyclonal antibodies commercially generated using

recombinant HDGF protein or goat pAb from R&D Systems), α-SMA (1:1,000 dilution,

clone 1A4, Sigma, St. Louis), pro-collagen α1 (I) (1:800 dilution, Santa Cruz

Biotechnology, Santa Cruz, CA), TGF-β (1: 4,500 dilution, MAB1032, Chemicon), and

β-actin (1:1,200 dilution, Santa Cruz), respectively. After five washes with PBS-T, the

blots were incubated with secondary antibodies, which were anti-mouse, -rabbit or

-goat immunoglobulin G coupled with horseradish peroxidase (HRP) enzyme and

pre-diluted at 1:15,000 to 1:20,000 in 5% non-fat milk of PBST buffer. After four

washes, the enhanced chemiluminescence (Santa Cruz ) detection system and x-ray

Max film (Amersham) were used to visualize the presence of immunoreactive proteins

on the blots according to the manufacturer’s instructions. The exposure time for each

protein (ranging from 1 min to 30 min) was calibrated by documenting linearity and

permitting semi-quantitative analysis. The visualized films were recorded on a digital

imaging system (UVI Tech, Cambridge, UK) and analyzed in a densitometrical analysis

- - 35

system (UVI Tech). Relative protein levels were expressed as the ratios of densities

between interested proteins and actin in the same sample.

Histopathological Examination

Sections were cut at a thickness of 4 µm and stained regularly with hematoxylin

and eosin (H&E). In brief, liver sections were deparaffinized in xylene and rehydrated

in a series of graded alcohol. After washing with distilled water for 10 min, Sections

were stained with hematoxylin for 2 mins for nuclear visualization. Then sections were

immersed in distilled water with 10 µl ammonia for 1 min and stained with eosin for 40

s. Finally, slides were dehydrated in alcohol, cleared in xylene, and mounted in

xylene-based mounting medium with coverslips. Histomorphology of liver section was

observed and documented with a light microscope (BX50, Olympus Optical Company,

Japan).

Sirius Red Stain and Histomorphometry

Sirius red stain was used for visualizing the intrahepatically deposited collagen

fibers. For Sirius red stain, formalin-fixed and paraffin-embedded sections were

deparaffinized with xylene and rehydrated with graded ethanol. After rinsing with

distilled water for 10 min, sections were stained with 0.1% Sirius red (Direct Red 80,

- - 36

Sigma) in saturated picric acid solution (Sigma) for 1 hr, followed by washing with two

changes of acidified water (0.5% acetic acid) and dehydration. The sections were

cleared in xylene and mounted. Morphology of collagen fibers was documented with a

light microscope (Olympus) equipped with CCD digital camera (DP-70, Olympus).

For histomorphometrical analysis, five randomly captured low-power images in

each section underwent morphometrical analysis using image analysis software

(Image-Pro Plus, ver. 6.1, Media Cybernetrics, Inc., Bethesda, MD). The percentage of

collagen deposition area was calculated with the function of collagenous area divided

by total image area.

Immunohistochemistry and Immunocytofluorescent Stain

Immunostaining of mouse liver slices was performed using the method with minor

modifications as previously described (Hu et al., 2003). Paraffin-embedded, 4 µm-thick

tissue sections were deparaffinized with xylene and rehydrated through decreasing

concentrations of alcohol. For antigen retrieval, tissue sections were heated in a

microwave oven at medium power in 0.01 mM citric buffer (pH6.0) for 15 minutes, and

then treated in 3% H2O2 for 10 minutes to quench endogenous peroxidase activity.

Staining was performed using an non-biotin in situ antigen detection kit (Polymer-HRP

detection IHC detection kit, BioGenex, San Ramon, CA) according to manufacturer’s

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instructions. Non-specific background was eliminated using serum blocking solution for

10 min. After washes with PBS, sections were incubated with rabbit anti-HDGF

polyclonal antibodies (1:200 dilution in PBS containing 1% bovine serum albumin and

0.1% Triton X-100) at 4 °C overnight. Then sections were incubated sequentially with

super enhancer solution and polymer-HRP conjugates. The peroxidase activity was

detected with DAB (Sigma), resulting in formation of brownish precipitates. Finally,

sections were briefly counterstained with hematoxylin for 10 sec, then dehydrated,

cleared and mounted in neutral gum under cover slips. For negative controls, the

fibrotic liver sections was applied with pre-immunized rabbit serum at equimolar

concentration.

For immunofluorescent staining, cultured cells were plated on collagen-coated

coverslips and fixed with 4% paraformaldehyde at 24h after gene delivery or

recombinant HDGF treatment. Anti-HDGF rabbit polyclonal antibody or

anti-polyHistidine mAb (R&D Systems) were used as primary antibody, followed by

visualization with Alexa488-conjugated goat anti-rabbit and Alexa594-conjugated goat

anti-mouse antibodies, respectively. Hoechst33342 were used as counter staining to

localize cell nuclei.

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TGF-β1 ELISA Assay

TGF-β1 levels in both mouse sera and culture conditioned media were determined

by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit

(Biosource, CA) according to manufacturer’s instruction. A monoclonal antibody

recognizing multispecies, including human, rat, and mouse TGF-β1, has been

pre-coated onto a microplate. Briefly, 200 µl of standards or samples and 50 µl of

biotinylated anti-TGF-β1 antibodies were simultaneously added into each well and

incubated for 3 hours at room temperature. After washes with washing buffer provided,

100 µl of streptavidin-HRP solution was added to each well and incubated 30 min at

room temperature. After washes, 100 µl of stabilized chromogen solution was added to

each well and incubated for 30 min at room temperature in the dark. Finally, the color

development was stopped by adding 100 µl of 1 M H2SO4 solution and the absorbance

was measured using a microplate reader (MRX II, Dynex technologies) at 450 nm.

Cell Culture

A normal rat hepatocyte cell line (Clone-9) derived from a normal

Sprague-Dawley rat liver was purchased from Bioresource Collection and Research

Center (BCRC no. 60201, Hsin-Chu, Taiwan) and a hepatic stellate cell line (clone

HSC-T6) was used for in vitro experiments. The cell lines were maintained in F-12K

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(Invitrogen) and Waymount’s (Sigma) media, respectively, both of which were

supplemented with 10% heat-inactivated fetal calf serum (FCS; Hyclone, Logan, UT),

penicillin and streptomycin (Invitrogen). Cells were incubated at 37 ºC in a humidified

atmosphere of 5 % CO2 in air, and the nutrient medium was renewed twice a week. For

adenoviral infection and cytokine stimulation experiments, the cells were replated at a

density of 5×105 cells/well onto 6-well plate pre-coated with or without collagen type I

extracts purified from rat tails.

Cell Proliferation Assays

Proliferation of normal and HDGF-treated or Ad-HDGF transfected cells was

determined using a cell-based 5-bromo-2’-deoxyuridine (BrdU) proliferation assay

(Roche Applied Science, Penzberg, Germany). Cells were allowed to attach for 24h

before exposure to HDGF or transfection with Ad-HDGF for 24h. Four hours before the

end of incubation, cells were labelled with 10 µM BrdU allowing for BrdU uptaking for

4h. Following fixation and denaturation, BrdU incorporation was immunodetected with

a peroxidase-conjugated monoclonal antibody directed against BrdU. Color

development was performed by adding 3,3’,5,5’,-tetramethyl benzidine (TMB) as

peroxidase substrate. The optical density was measured using a microplate reader

(MRX II, Dynex technologies) at 450 nm and control levels was considered as 100% of

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cell proliferation.

Flow Cytometry Analysis

HDGF-treated HSC-T6 cells (1×106) were harvested by trypsinization and

centrifugation. After being washed with PBS, the cells were incubated with 50 µl of

mouse anti-α-SMA antibodies (Sigma) with 1:200 dilution on ice for 1h. After washes

with PBS, the immunoreactive signal was visualized by secondary FITC-conjugated

goat anti-rabbit IgG antibody (Chemicon, Temecula, CA) at 1:1,000 dilution and

incubated on ice for another 1h. After washes the cells were fixed by 0.5%

paraformaldehyde in PBS for 10 min and then passed through a 30 µm mesh and for

subsequent FACScan detection (Elite ESP, Beckman Coulter, Brea, CA). The mean

fluorescent intensity (MFI) and the rate of α-SMA-positive cells in each group were

analyzed using software WinMDI (ver. 2.9, Windows Multiple Document Interface,

Flow Cytometry Application).

Gelatin Zymography

To determine the gelatinolytical activities of both MMP-2 and MMP-9 in the

conditioned media from HDGF-treated HSC T6 cells, SDS-PAGE for gelatin

zymography was performed using a previously reported method. Briefly, 10-15 µl of

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conditioned serum-free medium was used, and the volume was adjusted to contain the

same quantity of protein (5 µg). The medium supernatants were treated with

SDS-PAGE sample buffer without boiling and existence of reducing agent,

2-mercaptoethanol (2-ME). Samples were fractionated in a 8 % polyacrylamide gel

containing gelatin (2 mg/ml) by electrophoresis at 100 V for 90 min at 4℃. Afterwards,

acrylamide gels were soaked in 0.25 % Triton X-100 for 30 min at room temperature to

remove the SDS, and incubated in a digestion buffer (50 mM Tris-HCl, pH 7.4, 150

mM NaCl, 10 mM CaCl2, 2 µM ZnSO4 and 0.01 % Brij-35) containing 5 mM

phenyl-methylsulfonyl fluoride (PMSF), a serine protease inhibitor, at 37℃ overnight

to allow MMPs to digest its substrate gelatin in gel. Gels were rinsed with distilled

water, followed by staining with 0.25 % Coomassie brilliant blue R-250 in 50 %

methanol and 10 % acetic acid for 1 h and subsequent destaining with 10 % methanol

and 10% acetic acid. The gelatinolytic activities appeared as clear bands of digested

gelatin against a dark blue background of stained gelatin and were determined by

densitometrically measuring the band density.

Statistical Analysis

In vivo data were presented as mean ± standard error (SE), while in vitro as mean ±

standard deviation (SD). Difference among groups was statistically analyzed by

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one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons.

Significance is declared when P value is less than 0.05.

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Results

Liver fibrosis is reproducibly induced by BDL surgery and CCl4

administration.

To establish feasible animal models of liver fibrosis, two kinds of liver

fibrosis-inducing methods, i.e., BDL surgery and CCl4 chemical administration,

were conducted in ICR adult male mice as described in methodology section.

The mice sera and liver tissues in both models were collected at indicated time

points for measurement of biochemical parameters and fibrotic markers,

respectively.

As shown in Table 1, the biochemical analyses revealed that AST, ALT,

and LDH levels in sera of BDL-treated mice were significantly higher than those

in naïve control (P<0.05), and the levels peaked at post-operation day 7 (POD7),

but not at POD14. ALP levels were not completely done in this set of mice,

while γ-GT levels appeared increasing but beyond statistics. Intriguingly, TB and

DB levels in sera abruptly increased after BDL surgery (at both time points,

PODs 7 and 14) and both could be considered as the most reliable markers for

cholestasis in BDL model. The reliability is largely due to the severe cholestasis

resulting from surgically mimicked bile duct obstruction. On the other hand,

qRT-PCR analyses showed that the gene expression of collagen type I, TGF-β1,

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α-SMA significantly elevated along the progression of liver fibrosis (Fig. 3). In

parallel, the data of Western blotting detection and subsequent densitometrical

analysis showed significant increases in all three markers except for α-SMA

(Fig. 5), while the increasing trends were similar to those of corresponding

genes.

In comparison to BDL model, levels of AST, TB, and DB in mouse sera of

CCl4 model did not significantly change, only significant elevation of ALT and

LDH levels was noted at both day 18 and day 35 after CCl4 administration

(Table 2). Analysis of qRT-PCR showed that intraheptic gene transcripts for

collagen type I and TGF-β1 significantly increased after 18 days of CCl4

administration except that the significant induction of α-SMA gene was noticed

after 35 days of chemical treatment (Fig. 4). Similarly, translational levels of

collagen type I and TGF-β1 significantly increased after 18 days of CCl4

administration, whereas that of α-SMA slightly, but not significantly elevated as

detected by Western blotting (Fig. 6).

In terms of histopathological inspection, H&E staining and microscopic

examination indicated that portal fibrosis was prominently observed in portal

triad area in mice livers at 7 days after BDL surgery (Fig. 7B). In the meanwhile,

sparse distribution of both apoptotic and necrotic regions was characteristically

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and frequently noticed in BDL-induced fibrotic livers (Fig. 7C), while infiltration

of inflammatory cells such as neutrophils could be identified under higher

magnifications (Fig. 7D). In contrast, only the characteristic of portal fibrosis,

but not apoptotic and/or necrotic regions livers were seen in fibrotic livers under

microscope after 18 days of CCl4 administration (Fig. 7E).

To examine the distributional patterns and to further quantify the area of

collageneous matrix deposited , the intraheptic collagen matrix, primarily type I

collagen, was visualized by using Sirius red staining method. The microscopic

documentation clearly showed that only scanty of collagen fibers was seen

around basal membranes of hepatic arterioles, portal and central veins in normal

liver (Fig. 8A). At 14 days after BDL surgery, prominent accumulation of

fibrous collagen protruding from vascular basal membranes was noticed (Fig. 8B,

8C). In contrast to BDL model, the characteristic portal-to-portal bridging

network of collagen fiber deposition is formed and seen in CCl4-induced fibrotic

livers (Fig. 8D, 8E).

The above-mentioned results clearly manifested the reproducibility of liver

fibrosis induction using these two distinct methods. Accordingly, expression

profiles of HDGF in both models were next to be determined.

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HDGF expression is upregulated in fibrotic livers.

To determine the expression profiles of both HDGF gene and protein

during liver fibrosis, qRT-PCR and Western blotting analyses were, thus,

performed for the quantitative measurements. The gene expression of HDGF in

BDL model was significantly induced and the means of induction folds of HDGF

gene at POD 7 and POD 14 were 3.1 and 3.4, respectively, when normalized and

compared to naïve controls (Fig. 3). In contrast, the gene induction folds of

HDGF in CCl4 model was dramatically elevated up to 21 folds of controls for 18

days of chemical treatment and remained at this high level throughout the

experiment (Fig. 4). Consistent with qRT-PCR results, Western blotting

detection demonstrated that HDGF protein levels significantly increased along

with the progression of liver fibrosis induced by BDL surgery (Fig. 5) as well as

by CCl4 administration (Fig. 6). The elevated HDGF expression largely

displayed a time-dependent correlation with the expression levels of other

fibrosis-related markers, such as TGF-β1 and collagen type I. This result

implicates that HDGF may functionally relate with the expression of TGF-β1,

thereby subsequently affecting expression of collagen type I.

- - 47

HDGF is highly expressed in cytoplasm of perivasular hepatocytes.

To specify the cell type responsible for the HDGF upregulation,

immunohistochemistry was used to localize the HDGF protein expression. The

HDGF immunoreactivity was scarcely seen in parenchymal regions of normal

liver (Fig. 9A, 9B), but faintly found in nuclei of sinusoidal endothelial cells and

venous smooth muscle cells (data not shown). Intriguingly, a unique patched

staining pattern of HDGF was remarkably noticed in cytoplasmic compartment

of parenchyma sporadically surrounding lobular vasculatures of BDL

treatment-induced fibrotic livers (Fig. 9C, 9D). Similarly, the increased HDGF

signals in CCl4-injured livers were noticed in cytoplasm of perivenous

parenchymal cells (Fig. 9E) as well as in those distributed along fibrous septa

(Fig. 9F). These findings clearly indicate that the upregulated HDGF expression

during liver fibrosis is mainly localized in cytoplasm of perivascular hepatocytes

and suggest that the upregulated HDGF protein may release and stimulate other

component cells in liver in a paracrinal manner.

HDGF gene delivery alone deteriorates functional integrity of liver.

To evaluate the effect of HDGF overexpression on hepatocellular integrity

and functions, mice serum and liver tissues were monitored at 14 days after

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adenovirus-mediated gene delivery. Table 3 indicated that, among biochemical

parameters, both serum AST and ALP levels in Ad-HDGF-infected mice were

significantly higher than those in naïve control and Ad-GFP-infected group (Fig.

11A), suggesting that HDGF gene delivery per se leads to functional

deterioration of normal liver. Histological examination showed that 66.6% (4 out

of 6) of mice in Ad-HDGF group, but only 16.6% (1 out of 6) of mice in Ad-GFP

group, displayed typical cytoplasmic vacuolization as revealed by H&E staining

and microscopic observation (Fig. 10).

To visualize the distribution of collagen bundles, Sirius red staining was

performed and followed by histomorphometrical analysis. The staining result

indicated that Ad-HDGF gene delivery appeared to thicken the collagen bundles

beneath endothelium (Fig. 10). Besides, the histomorphometrical analysis

showed that Ad-HDGF gene delivery significantly enhanced the deposition of

collagen fiber as compared to that in naïve livers, although marginally differed

from that in Ad-GFP group (Fig. 11B).

HDGF gene delivery aggravates liver fibrosis.

To further investigate whether HDGF overexpression enhances severity of

liver fibrosis, adenoviral gene delivery was utilized one day before

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implementation of BDL and CCl4 administration. Serum and liver tissues of

BDL- and CCl4-treated mice were harvested at 14 and 35 days, respectively, and

underwent biochemical analysis and molecular detection.

The Biochemical data shown in Table 4 indicated that both Ad-GFP and

Ad-HDGF gene deliveries potentiated the BDL-induced hepatic malfunction in

terms of serum AST and ALT levels, but no statistical significance was obtained

between Ad-GFP- and Ad-HDGF-pretreated groups.

Similarly, the biochemical parameters measured in CCl4 model showed no

consistency in that only the mean of AST levels in Ad-HDGF was elevated

higher than, but not significantly, that in Ad-GFP, while both of ALT and ALP

levels in Ad-HDGF-treated group were, on the contrary, lower than those in

Ad-GFP vehicle control (Table 5). The discrepancy may be due to the possibility

that Ad-HDGF-mediated HDGF overexpression may also contribute to the

regenerative process of chronically injured livers. In parallel, the TGF-β1 levels

in sera of normal and adenovirally infected mice also showed no consistency and

no conclusion was obtainable (Table 6).

However, Western blotting analysis demonstrated that Ad-HDGF gene

delivery significantly potentiated the increased levels of TGF-β1 and

pro-collagen α1(I) protein in mice with BDL surgery (Fig. 12) as well as with

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CCl4 administration (Fig. 13) as compared to vehicle control.

To scrutinize the effect of Ad-HDGF gene delivery on intrahepatic

deposition of collagen fibers, Sirius red stain (Fig. 14) followed by

histomorphometrical analysis (Fig. 15) were performed. The results

demonstrated that the deposited area of intrahepatic collagen fibers increased

along with the progression of liver fibrosis in mice with BDL (Fig. 15A) and

CCl4 (Fig. 15C) treatment. Whereas Ad-HDGF gene delivery significantly

potentiated the collagen deposition therein (Fig. 15B, D), as compared to

corresponding Ad-GFP-treated groups.

A unique mutual regulation between HDGF and TGF-ββββ1111 expression in

hepatocytes.

Given that a basement membrane structure is newly formed de novo in the

space of Disse during the progression of liver fibrosis (Quondamatteo et al.,

2004), and that collagen I is constitutively localized both in areas around

vascular network and peri-sinusoidal regions in livers as revealed by

ultrastructural analyses (Pinkse et al., 2004), we hypothesized that cell-ECM

interaction might account for the differential responsiveness of hepatocytes to

pro-fibrogenic stimuli.

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To elucidate the mechanism through which HDGF predominantly expresses

in perivascular hepatocytes, a cell line of hepatocytes (clone-9 cells derived from

a SD rat) grown on either collagen I-coated or -uncoated dishes were treated with

recombinant TGF-β1, the most potent fibrogenic factor, and the total RNA and

protein were collected for further analyses of HDGF gene and protein expression.

In accordance with our expectation based on in vivo observation, exogenous

treatment with TGF-β1 resulted in a collagen I-dependent increase of HDGF

expression at both transcriptional (Fig. 16) and translational (Fig. 17) levels in

cultured hepatocytes.

To examine the impact of HDGF overexpression in hepatocytes on

fibrogenesis, the cultured hepatocytes received with adenovirally-induced HDGF

overexpression for 48 h were collected for measuring the contents of

pro-collagen I in lysates as well as intra- and extracellular HDGF protein. The

Western blotting result clearly showed that Ad-HDGF gene delivery not only led

to an elevation of HDGF protein expression in cultured hepatocytes, but also an

increase in the biosynthesis of type I collagen pro-peptides, in concomitance with

a remarkable soluble HDGF release from cultured hepatocytes (Fig. 18). This

result implicates that the overexpressed HDGF protein in perivascular

hepatocytes may substantially contribute to the biosynthesis of type I collagen in

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hepatocytes themselves. In addition, it also raises the possibilities that the

overexpressed HDGF in hepatocytes may autoregulate TGF-β1 expression in

themselves, and that the soluble HDGF peptide released from hepatocytes may

stimulate HSCs in a paracrinal manner.

To determine whether HDGF treatment could exogenously upregulate de

novo synthesis of TGF-β1 in cultured hepatocytes, clone-9 hepatocytes grown on

either collagen I-coated or -uncoated dishes were treated with recombinant

HDGF and the total RNA and protein were collected for qRT-PCR and ELISA

detection. The results indicated that the treatment with exogenous HDGF,

irrespective of pre-coating with collagen I or not, significantly up-regulate

TGF-β1 expression in cultured hepatocytes, as evidenced by qRT-PCR (Fig. 19A)

as well as by ELISA (Fig. 19B). This result clearly points out that a mutual

regulation uniquely exists between the de novo syntheses of HDGF and TGF-β1.

Through such a vicious circle, HDGF may pathogenically amplify the

TGF-β1-triggered pro-fibrogenic signaling during the development of liver

fibrosis. This is possibly the pro-fibrogenic role played by HDGF.

Exogenous HDGF enhances ECM and αααα-SMA expression in HSCs.

Because the soluble HDGF released from HDGF-overexpressing

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hepatocytes might activate HSCs in a paracrinal manner, we sought to

demonstrate this speculation. To examine whether exogenous HDGF exhibits

fibroproliferative effects on HSC, a cell line of HSCs (HSC-T6 cells), which

have been preliminarily characterized as an activated phenotype expressing

α-SMA (Fig. 20), were serum-starved for overnight and treated with

recombinant HDGF in the presence of 0.5% FBS. The uptake of recombinant

His-tagged HDGF peptide was predominantly localized on membrane or in

cytoplasm of cultured HSCs as visualized by immunofluorescent cytostaining

(Fig. 21). Stimulation with exogenous HDGF at concentrations higher than 0.1

ng/ml significantly raised BrdU incorporation rate in cultured HSCs, while

treatment at 10 ng/ml resulted in a 70% increase in BrdU uptake as compared

with control levels (Fig. 22). Concomitantly, exogenous HDGF prominently

increased α-SMA and collagen type I biosynthesis at both transcriptional and

translational levels, as evidenced by qRT-PCR analyses (Fig. 23) and Western

blotting (Fig. 24). In parallel, using flow cytometrical method we confirmed

again that exogenous HDGF upregulated α-SMA expression in cultured HSCs in

the presence of 10% FBS (Fig. 25).

To further explore the fibroproliferative effect of endogenous HDGF on

expansion of HSC population, cell proliferation and BrdU assays were

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performed after Ad-HDGF gene delivery in vitro. Ad-HDGF infection at dose of

MOI50 for 48 h induced a dense HDGF immunoreactivity in nuclei of HSCs, as

compared to the equally distributed staining pattern seen in Ad-GFP control

group (Fig. 26). Meanwhile, Ad-HDGF gene delivery induced a minor, but

statistically significant increase by 30% of control levels in both cell

proliferation (Fig. 27) and BrdU assays (Fig. 28). On the contrary, Ad-HDGF

gene delivery did not affect the production of pro-collagen type I and α-SMA

proteins (Fig. 29). These results collectively indicated that nuclear localization of

HDGF protein may only contribute to mitogenesis of HSCs, instead of

transdifferentiation or cellular transformation.

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Discussion

The present study provides novel insights into the mechanism that regulate

intrahepatic TGF-β1 expression and fibrogenesis. Although Yoshida et al have

speculated that HDGF expression might be involved in hepatic fibrogenesis

before tumor development (Yoshida et al., 2003), we are the first to present the

supportive evidence based on the data from experimental models of liver fibrosis.

The sketch of the possible role of HDGF in hepatic fibrogenesis is shown and

described in Figure 30.

In this study, we demonstrate that HDGF overexpression is intimately

involved in the pathogenesis of liver fibrosis, by unraveling the temporal

accordance between the expression of intrahepatic HDGF and fibrotic markers,

such as TGF-β1 and pro-collagen type I, as well as the progression of liver

fibrosis. The unique staining pattern of HDGF immunohistochemistry clearly

indicates that the overexpressed HDGF in fibrotic livers is mainly localized in

the cytoplasmic compartment of perivascular hepatocytes. Pretreatment with

HDGF gene vector promotes the synthesis of TGF-β1 and pro-collagen type I,

leading to enhanced collageneous matrix deposition in liver. Besides, in vitro

evidence shows that the mutual upregulation existing between HDGF and

TGF-β1 expression in the cultured hepatocytes grown on collagenous matrix

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may facilitate the induction of hepatic fibrogenesis. In vitro HDGF gene delivery

results in soluble HDGF peptide release from hepatocytes, while exogenous

HDGF administration enhances both mitogenesis and phenotypic transformation

of cultured HSCs, strongly suggesting that the paracrinal action mode of HDGF

between these two cell types may underlie the pathogenesis of fibrotic liver

diseases.

In the context of biological functions, HDGF has been demonstrated to

reside in both cytoplasm and nucleus according to cell type and phase of cell

cycle (Everett et al., 2000; Everett et al., 2004; Mori et al., 2004; Zhang et al.,

2006a), whereas the specific functions of HDGF in both compartments remain

mysterious. The nuclear localization of HDGF in many tumor cells suggests its

mitogenic role, although cytoplasmic HDGF accumulation in hepatic and gastric

carcinoma also reveals prognostic significance (Everett et al., 2000; Yamamoto

et al., 2006). The pleiotropic characteristic of HDGF implicates its role in tissue

homeostasis, including hepatic fibrogenesis. As a matter of fact, in addition to

serving as an intracrine signaling factor, HDGF is capable of being

extracellularly released to regulate growth of surrounding cells. The HDGF

overexpression in necrotic neurons has been demonstrated to serve as a

neurotrophic factor that rescues neuronal cell death when exogenously supplied

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(Zhou et al., 2004). Besides, HDGF overexpression in fibrotic lungs is found to

positively regulate lung epithelial cell proliferation (Mori et al., 2004). Based

upon the significant correlation between HDGF expression and hepatic

fibrogenesis found in this study, we reason that HDGF is constitutively expressed

at low levels in parenchyma of normal adult liver, while HDGF expression and

its release may be either autonomously regulated under stresses or responsively

induced by other pathogenetic cytokines.

The present study identifies that parenchymal hepatocyte is the major cell

type expressing HDGF, as evidenced by immunohistochemistry in both models

of liver fibrosis. The cytoplasmic localization suggests that the paracrinal effects

of HDGF on surrounding nonparenchymal cells may outweigh the autocrinal or

intracrinal functions in regulating hepatocyte proliferation per se. To support this,

the adenoviral HDGF delivery induces not only its overexpression in but also

extracellular release from cultured hepatocytes (Fig. 18), while the uptake of

exogenously supplied recombinant HDGF by HSCs raises the possibility that the

elevated HDGF in hepatocytes might be released, thus in a paracrine manner

stimulate HSC proliferation and enhance the transdifferentiation of HSCs with

simultaneously upregulating α-SMA expression (Figs. 23,24). Moreover,

adenoviral HDGF gene delivery aggravates the collageneous matrix deposition

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in fibrotic livers (Figs. 12-15).

The HDGF expression levels in hepatoma and its nuclear localization have

been previously demonstrated to be negatively proportional to and intimately

correlated with the degree of cell differentiation (Hu et al., 2003). In contrast, the

moderate levels of HDGF expression and its cytoplasmic localization in fibrotic

livers indicate that parenchymal hepatocytes therein have not yet been

transformed to neoplastic phenotype. Besides, HDGF is known to be involved in

liver development and regeneration (Enomoto et al., 2002a; Enomoto et al.,

2002b), both require formation of vascular microarchitecture to supply

parenchymal nutrition. To support this, the pro-angiogenic role of HDGF is

unraveled by the evidence that HDGF is a pulmonary endothelial cell-expressing

angiogenic factor (Everett et al., 2004) and it can directly induce VEGF

expression in tumors (Okuda et al., 2003). We previously also observed a

positive correlation between cytoplasmic HDGF and VEGF expression in HCC

specimens (Hu et al., 2003), and the regulatory mechanism may involve the

activation of NF-κB signaling pathway (unpublished data). The interactions

between VEGF and its cognate receptors are found to be essential for the

development of liver fibrosis in animals (Corpechot et al., 2002; Yoshiji et al.,

2003). An unusually persistent activation of NF-κB seen in activated HSCs

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suggests that HDGF may also activate HSCs via NF-κB pathway (Novo et al.,

2007). Taken together, the upregulated HDGF in livers may not only promote

HSC activation but also participate in neoangiogenesis and sinusoidal

capillarization in chronic liver diseases, thus leading to liver fibrosis and

eventually tumorigenesis and metastasis.

A plethora of cytokines induced and their interaction between parenchymal

and nonparenchymal liver cells have been suggested to contribute to hepatic

fibrogenesis (Ramm et al., 1998; Bataller and Brenner, 2005; Tsukada et al.,

2006). In this regard, sustained overexpression of TGF-β1, a hallmark for

fibrotic disease in all tissues, is abundantly found in both constituents (De Bleser

et al., 1997). However, the underlying mechanism governing TGF-β1 synthesis

in liver constituent cells remains ambiguous. The data in this study clearly

displayed that HDGF expression in cultured hepatocytes is upregulated by

TGF-β1 in a collagen-dependent manner (Figs. 16,17). Combined with the

evidence that TGF-β1 expresses in hepatocytes proximal to fibrotic areas at

interface of regenerative nodule (De Bleser et al., 1997; Ramm et al., 1998) and

that the activated macrophages aggregate within HDGF-highly expressing foci

(Yoshida et al., 2003), our data etiologically propose that TGF-β1 derived from

infiltrating immune cells or Kupffer cells may induce HDGF expression in

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proximal hepatic parenchyma. Either the early emergence of TGF-β1 during

acute hepatic inflammation or the hypoxia-induced TGF-β1 elevation in

chronically-diseased livers may initiate the de novo synthesis of HDGF, and vice

versa. The ECM-dependent mutual regulation between TGF-β1 and HDGF

expression highlights the pro-fibrogenic role of HDGF (Figs. 16,17,19), further

strengthening the notion that elevation and secretion of HDGF may contribute

either to augmentation of TGF-β synthesis, direct activation of HSCs, or

potentiated responsiveness of surrounding HSCs to TGF-β stimulus, thereby

accelerating the progression of liver fibrogenesis via such a vicious circle.

The molecular mechanism by which HDGF regulates TGF-β synthesis

remains poorly defined. To date, no direct evidence indicates how HDGF

upregulates de novo synthesis of TGF-β. However, the fact that HDGF activates

NF-κB signaling supports the result found in this study because the

NF-κB-dependent TGF-β production has been evidenced in many phenotypes of

cells (Rameshwar et al., 2000; Lawrence et al., 2001; Komura et al., 2005).

HDGF is also likely to mediate TGF-β synthesis through an NF-kB-dependent

pathway, while this hypothesis awaits further exploitation. In the aspect of how

TGF-β upregulates HDGF, to date it remains a mystery due to the scanty

information about the regulatory element in HDGF promoter site. More work is

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needed to delineate the possible regulatory mechanism.

The excessive deposition of ECM proteins as such not only disrupts normal

microarchitecture and function of liver organ, but also affects the responsiveness

of hepatocytes to external stimuli. A similar finding in renal fibrosis points out

that collagen I modulate TGF-β1 secretion and ECM expression in mesangial

cells (Ortega-Velazquez et al., 2004). In this context, ECM is known to convey

specific signals to cells that act in concert with growth factors (Tai et al., 2003b;

Clemmons and Maile, 2005; Chan et al., 2006). The ECM receptors, integrins,

play a critical role in regulating diverse cellular functions including growth,

migration, differentiation, gene expression, and ECM remodeling (Larsen et al.,

2006; Luo and Springer, 2006; Moissoglu and Schwartz, 2006). Indeed,

elevation of integrin subunit β1 and other α subunits were remarkably noticed in

fibrotic livers (Garcia-Monzon et al., 1992; Scoazec, 1995; Quondamatteo et al.,

2004). The kinase mediators of integrin signaling cascade, including focal

adhesion kinase (von Sengbusch et al., 2005) and integrin-linked kinase (ILK)

(Abboud et al., 2007), are suggested to mediate hepatoma transformation and

metastasis via activation of protein kinase B signaling pathway (Xu et al., 2003).

Moreover, ILK deficiency results in prevention of TGF-β-mediated E-cadherin

delocalization (Lee et al., 2004). Conversely, hyperfunctioning of ILK has been

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observed in renal fibrosis (Li et al., 2003) and, more recently, in liver fibrosis

(Zhang et al., 2006b). Our data in this study suggest that the over-expressed

HDGF may intervene and potentiate the interactions among cytokines,

hepatocytes, and ECM molecules, possibly conveying either anti-apoptotic or

pro-fibrogenic signals, or both. However, the molecular mechanisms underlying

the two-way regulation between TGF-β and HDGF as well as the capacity of

HDGF to modulate the TGF-β receptors and relevant pro-fibrogenic signalings

in HSCs remain to be further explored.

In conclusion, the HDGF overexpression in fibrotic livers may contribute

to TGF-β upregulation in a collagen-dependent manner, while TGF-β mutually

stimulates HDGF synthesis in an autocrine manner. Through such a unique

action mode, HDGF amplifies the TGF-β-driven pro-fibrogenic signaling and

simultaneously promotes HSC activation in a paracrine manner during

progression of liver fibrosis. Therefore, blockade of HDGF pathway may

potentially constitute the preventive or therapeutic strategies for chronic liver

diseases.

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Future Perspective

Although in this study we have addressed both in vivo and in vitro evidence

supporting that HDGF plays a pro-fibrogenic role during hepatic fibrogenesis,

there still are several questions remained to be solved:

Firstly, to date no evidence has yet indicated that the HDGF expression

profile in human specimens of liver fibrosis shows the same trend as those in

animal models. To answer this question, clinical specimens from the Liver

Transplantation Laboratory in Kaohsiung Chang Gung Memorial Hospital, are

now in collection for further analysis.

Secondly, it is urgent to determine whether anti-HDGF interventions, such

as transfection with anti-sense viral vectors and/or small interfering RNA to

knock down HDGF gene transcript, could effectively serve as anti-fibrotic

strategies. To solve this problem, the investigation on the effectiveness of HDGF

siRNA in mice animal models is now in preparation. We believe that completion

of these two parts of surveys will help better understand the clinical applicability

of anti-HDGF strategy.

Furthermore, the other line of experiment has demonstrated that HDGF

possesses the capacities to directly suppress the gelatinolytic activity of MMP-2

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and to inhibit the TNF-α-induced MMP-2 upregulation (as shown in Fig. 31).

Clarification of the anti-gelatinolytic function of HDGF or the possible

intervention in activities of corresponding TIMPs may hopefully help elucidate

the underlying molecular mechanism(s) of HDGF in driving fibrogenesis.

- - 65

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Tables

Table 1. Biochemical analysis for sera of mice with BDL surgery.

Parameter (unit) Day 0 (n=6) Day 7 (n=5) Day 14 (n=5)

AST (U/L) 129.3 ± 6.9 1769.4 ± 525.1* 646.8 ± 78.5*

ALT (U/L) 32.8 ± 7.0 946.2 ± 326.3* 427.8 ± 37.8*

ALP (U/L) 105.8 ± 6.2 N.D. N.D.

T.B. (mg/dL) 0.17 ± 0.16 9.3 ± 1.3* 8.9 ± 0.5*

D.B. (mg/dL) 0 ± 0 7.7 ± 1.02* 7.7 ± 0.33*

LDH (U/L) 429.0 ± 87.3 1246.2 ± 277.8* 661.2 ± 20.7*

γ-GT (U/L) 0 ± 0 3 ± 0 3 ± 0

Data are expressed as mean ± SE.

AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP,

alkaline phosphotase; T.B., total bilirubin; D.B., direct bilirubin; LDH, lactate

dehydrogenase; γ-GT, gamma-glutamyltranspeptidase.

N.D., not done; * indicates P<0.05 compared with the control group at day 0.

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Table 2. Biochemical analysis for sera of mice with CCl4 administration. Parameter (unit) Day 0 (n=6) Day 18 (n=5) Day 35 (n=4)

AST (U/L) 129.3 ± 6.9 108.8 ± 29.3 136.0 ± 13.3

ALT (U/L) 32.8 ± 7.0 68.4 ± 7.6* 52.0 ± 2.0*

ALP (U/L) 105.8 ± 6.2 N.D. N.D.

T.B. (mg/dL) 0.17 ± 0.26 0.08 ± 0.05 0.06 ± 0.05

D.B. (mg/dL) 0 ± 0 0 ± 0 0 ± 0

LDH (U/L) 429.0 ± 87.3 640.0 ± 49.4* 511.5 ± 25.3*

γ-GT (U/L) 0 ± 0 0.02 ± 0 0.02 ± 0

Data are expressed as mean ± SE.

AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline

phosphotase; T.B., total bilirubin; D.B., direct bilirubin; LDH, lactate

dehydrogenase; γ-GT, gamma-glutamyltranspeptidase.

N.D., not done; * indicates P<0.05 compared with the control group at day 0.

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Table 3. Biochemical analysis for sera of mice with adenoviral gene delivery.

Parameter (unit) Naive (n=6) Ad-GFP (n=6) Ad-HDGF (n=6)

AST (U/L) 129.3 ± 6.9 118.3 ± 20.7* 197.5 ± 36.4*†

ALT (U/L) 32.8 ± 7.0 32.5 ± 2.8 32.5 ± 4.6

ALP (U/L) 105.8 ± 6.2 120.0 ± 10.9 136.7 ± 15.4*

T.B. (mg/dL) 0.17 ± 0.26 0.25 ± 0.27 0.17 ± 0.26

D.B. (mg/dL) 0 ± 0 0 ± 0 0 ± 0

Data are expressed as mean ± SE.

AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP,

alkaline phosphotase; T.B., total bilirubin; D.B., direct bilirubin.

* indicates P<0.05 compared with the naïve control group.

†: P<0.05 compared with the Ad-GFP group

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Table 4. Biochemical analysis for sera of mice with both adenovirus-mediated HDGF gene delivery and BDL surgery.

Parameter

(unit)

Naive

(n=6)

BDL alone

(n=5)

w/ Ad-GFP

(n=4)

w/ Ad-HDGF

(n=5)

AST (U/L) 129.3 ± 6.9 646.8 ± 78.5* 1221.3 ± 168.4*† 1772.0 ± 379.8.0*†

ALT (U/L) 32.8 ± 7.0 427.8 ± 37.8* 518.8 ± 56.5* 862.0 ± 192.9*†

ALP (U/L) 105.8 ± 6.2 N.D. 2023.8 ± 574.7* 1794.0 ± 228.7*

T.B. (mg/dL) 0.17 ± 0.10 8.9 ± 0.5* 14.8 ± 4.1* 12.0 ± 1.8*

D.B. (mg/dL) 0 ± 0 7.74 ± 0.33* 12.50 ± 3.30* 10.30 ± 1.50*

Data are expressed as mean ± SE.

AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline

phosphotase; T.B., total bilirubin; D.B., direct bilirubin.

N.D., not done

*: P<0.05 compared with the naïve control;

†: P<0.05 compared with the BDL alone (nil-adenoviral treatment) groups at day

14.

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Table 5. Biochemical analysis for sera of mice with both adenovirus-mediated HDGF gene delivery and CCl4 administration.

Parameter

(unit)

Naive

(n=6)

CCl4 alone

(n=4)

Ad-GFP

(n=3)

Ad-HDGF

(n=5)

AST (U/L) 129.3 ± 6.9 136.0 ± 26.7 228.3 ± 34.0*† 247.0 ± 155.9

ALT (U/L) 32.8 ± 7.0 52.0 ± 4.0 151.7 ± 7.6*† 108.0 ± 58.7*

ALP (U/L) 105.8 ± 6.2 N.D. 200.0 ± 72.1* 71.0 ± 18.5*‡

T.B. (mg/dL) 0.16 ± 0.10 0.06 ± 0.05 N.D. N.D.

D.B. (mg/dL) 0 ± 0 0 ± 0 0 ± 0 0 ± 0

Data are expressed as mean ± SE.

AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP,

alkaline phosphotase; T.B., total bilirubin; D.B., direct bilirubin.

N.D., not done;

*: P<0.05 compared with the naïve control;

†: P <0.05 compared with the CCl4 alone (nil-adenoviral treatment) groups at

day 35;

‡: P <0.05 compared with the Ad-GFP vehicle control groups.

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Table 6. TGF-β levels in sera of normal and adenovirally infected mice

Groups TGF-ββββ (pg/ml)

Control (n=6) 660.9 ± 18.9

Ad-GFP (n=4) 813.6 ± 52.9

Ad-HDGF (n=5) 732.8 ± 72.9 *

BDL+Ad-GFP (n=4) 1658.7 ± 167.7 *

BDL+Ad-HDGF (n=5) 876.7 ± 77.6 *,†

CCl4 +Ad-GFP (n=3) 530.1 ± 98.8

CCl4 +Ad-HDGF (n=4) 751.4 ± 165.3

Mice sera were collected at day14 for both adenoviral infection and

BDL-treated groups, and at day 35 for CCl4-treated groups. Data are expressed as mean ± SEM of triplicate tests for each

sample.

*: P <0.05 compared with the control group;

†: P <0.05 compared with the respective Ad-GFP vehicle control

group.

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Figures

A BDL model

CCl4 model

Day 18 Day 35Day 0

1 mL/Kg s.c., twice weekly

Day 7 Day 14Day 0

BDL surgery

B

BDL model

CCl4 model

Day 14Day 0

BDL surgery

Day 35Day 0

1 mL/Kg s.c., twice weekly

Figure 1. Flow chart of experimental mouse models for liver fibrosis induction.

(A) To characterize the expression profiles of HDGF and liver fibrosis-associated

markers, liver fibrosis was induced either surgically by bile duct ligation (BDL) or

chemically by subcutaneous administration of CCl4. Arrows indicate the time

points for s.c. injection at a dose of 1 mL/Kg twice weekly, while inverted solid

triangles indicate the time points for specimen collection.; (B) To determine the

effects of HDGF overexpression on the progression of liver fibrosis, the Ad-GFP

or Ad-HDGF diluted in saline was administered through the tail vein at a dose of

1×109 plaque-forming units each mouse, under ether anesthesia at 24 h prior to

the BDL surgery or the first CCl4 injection.

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Figure 2. Purification of recombinant human HDGF protein and identification

of its efficacy. Human HDGF cDNA, obtained from a human fetal brain cDNA

library, was subcloned into expression vector and the recombinant HDGF

protein was purified as described in METHODOLOGY section. (A) The

molecular weight was identified using reducing SDS-PAGE; (B) Dosages higher

than 10 ng/ml of HDGF significantly stimulate cell proliferation of NIH-3T3

fibroblasts, as evidenced by 3H-thymidine uptake. (C) Dosage higher than 10

ng/ml of HDGF significantly stimulates cell migration of NIH-3T3 fibroblasts. (*:P

<0.05, as compared to control) The data excerpted in this figure was kindly

provided by Dr. Tai, MH.

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Control d7 d14

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*

Figure 3. Increased expression of HDGF gene in mouse fibrotic livers induced

by BDL surgery. Liver tissues were collected from mice treated with BDL surgery

after 7 (d7) and 14 days (d14), respectively. qRT-PCR analyses showed that

HDGF gene is upregulated in both liver fibrosis models and correlated with the

progression of liver fibrosis. Data were expressed as mean induction fold

compared to control ± SE. *, P<0.05.

- - 89

Control d18 d35

HD

GF

gen

e ex

pre

ssio

n(i

nd

uct

ion

fo

lds

to c

on

tro

l)

0

5

10

15

20

25

30

(n=6) (n=5) (n=4)

**

Control d18 d35

αα αα-S

MA

gen

e ex

pre

ssio

n(i

nd

uct

ion

fo

lds

to c

on

tro

l)

0

1

2

3

(n=6) (n=5) (n=4)

*

Control d18 d35

TG

F- ββ ββ

gen

e ex

pre

ssio

n(i

nd

uct

ion

fo

lds

to c

on

tro

l)

0

2

4

6

8

10

12

(n=6) (n=5) (n=4)

**

Control d18 d35

Co

llag

en I

gen

e ex

pre

ssio

n(i

nd

uct

ion

fo

lds

to c

on

tro

l)

0

10

20

30

40

50

(n=6) (n=5) (n=4)

**

*

Figure 4. Increased expression of HDGF gene in mouse fibrotic livers induced

by CCl4 administration. Liver tissues were collected from mice treated with CCl4

administration after 18 (d18) and 35 days (d35), respectively. qRT-PCR

analyses showed that HDGF gene is upregulated in both liver fibrosis models

and correlated with the progression of liver fibrosis. Data were expressed as

mean induction fold compared to control ± SE. * , P<0.05; ** , P<0.01.

- - 90

A

Actin

1 2 1 2 3 4 5 1 2 3 4 5

Control d 7 d 14

HDGF

αααα-SMA

TGF-ββββ

Pro-collagen I

B

Control d7 d14

HD

GF

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

1

2

(n=6) (n=5) (n=5)

**

Control d7 d14

αα αα-S

MA

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

1

2

(n=6) (n=5) (n=5)

Control d7 d14

TG

F- ββ ββ

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

1

2

3

4

5

*

**

(n=6) (n=5) (n=5) Control d7 d14

Pro

-co

llag

en αα αα

1 (I

) ex

pre

ssio

n(i

nd

uct

ion

fo

lds

to c

on

tro

l)

0

1

2

3

4

5

6

(n=6) (n=5) (n=5)

**

**

Figure 5. Elevated HDGF protein levels in BDL-treated mouse fibrotic livers. (A) Liver tissues were collected from mice treated with BDL surgery after 7 (d7)

and 14 days (d14), respectively. Total protein extracts were subjected for

Western blotting analysis. Representative images for HDGF, α-SMA, TGF-β,

and procollagen α1 (I) were shown. (B) Densitometrical analysis for respective protein contents. Data are presented as mean induction fold to control ± SE. * ,

P<0.05; ** , P<0.01.

- - 91

A

HDGF

1 2 1 2 3 4 5 1 2 3 4

Control d 18 d 35

αααα-SMA

Pro-collagen I

TGF-ββββ

Actin

B

Control d18 d35

HD

GF

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

1

2

3

4

(n=6) (n=5) (n=4)

*

**

Control d18 d35

αα αα-S

MA

exp

ress

ion

(in

du

ctio

n f

old

s to

act

in)

0

1

2

(n=6) (n=5) (n=4)

Control d18 d35

TG

F- ββ ββ

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

1

2

3

4

5

6

(n=6) (n=5) (n=4)

****

Control d18 d35

Pro

-co

llag

en αα αα

1 (I

) ex

pre

ssio

n(i

nd

uct

ion

fo

lds

to c

on

tro

l)

0

2

4

6

8

(n=6) (n=5) (n=4)

**

**

Figure 6. Elevated HDGF protein expression in CCl4-induced fibrotic liver tissues of mice. (A) Liver tissues were collected from mice treated with CCl4

administration after 18 (d18) and 35 days (d35), respectively. Total protein

extracts were used for Western blotting analysis. Representative images for

HDGF, α-SMA, TGF-β, and procollagen α1 (I) were shown. (B) Densitometrical

analysis for respective protein contents. Data are presented as mean induction fold to control ± SE. * , P<0.05; ** , P<0.01.

- - 92

Figure 7. Microscopic examination on histopathological alteration in fibrotic livers. Paraffin-embedded liver tissues were sectioned and stained with H&E,

followed by microscopic documentation. (A) Normal liver; (B) BDL-induced

fibrotic liver (at day 7); (C) apoptotic and necrotic regions were frequently noted

in BDL-fibrotic livers; (D) infiltration of inflammatory cells in necrotic and

apoptotic region; (E) histological observation in CCl4-induced fibrotic livers (at

day 35). Scale bar = 200 µm.

A B

C D

E

- - 93

Figure 8. Visualization of intrahepatic deposition of fibrous collagen. Paraffin-embedded liver tissues were sectioned and stained with H&E, followed

by microscopic documentation. Scanty collagen deposition on vascular basal

membrane was noted in normal liver (A). Fibrous collagen accumulates around

and protrude from vascular basal membrane in BDL-induced fibrotic liver (at day

14) (B, C); Characteristic portal-portal bridging network of collagen fiber

deposition is formed and seen in CCl4-induced fibrotic livers (at day 35) (D, E).

Scale bar = 200 µm.

A

B C

D E

- - 94

A

B

C

D

E

F

Figure 9. Upregulated HDGF expression in fibrotic livers is predominantly localized in perivascular hepatocytes. Seven days and 18 days after induction of liver fibrosis with BDL or CCl4 treatments, respectively, immunohistochemistry was performed to detect the HDGF localization. Representative microphotographs for HDGF expression in normal livers (A, B) and fibrotic livers with BDL (C, D) and CCl4 (E, F) treatment. Scale bar = 100 µm.

- - 95

Figure 10. HDGF gene delivery leads to hepatocellular injury. Liver sections

were obtained at 14 days after adenovirus administration and underwent H&E

stain (left panel). Sections in 66.6% (4 out of 6) rats in Ad-HDGF group showed

the typical cytoplasmic vacuolization and hepatocellular injury. Collagen fibers in

liver sections were visualized using Sirius red staining and representative

microphotographs were shown (right panel). Insets, higher magnificance. Scale

Bar, 200 µm.

Control

Ad-GFP

Ad-HDGF

Control

Ad-GFP

Ad-HDGF

- - 96

A B

Control Ad-GFP Ad-HDGF

AS

T le

vels

(U

/L)

50

100

150

200

250 **

(n=6) (n=6) (n=6)

% A

rea

of

Co

llag

en D

epo

siti

on

0

1

*

Control Ad-GFP Ad-HDGF(n=6) (n=6) (n=6)

*

Figure 11. HDGF gene delivery leads to hepatocellular injury. (A) After being

infected with adenoviruses for 14 days, mouse sera were collected for

hepatocellular enzyme measurements, including AST. (B) Liver sections were

obtained at 14 days after adenovirus administration and underwent Sirius red

staining. Morphometrical analyses for area of collagen deposition showed that

HDGF gene delivery exhibits pro-fibrogenic propensity. Data are presented as

mean ± SE. * , P<0.05.

- - 97

A

ββββ-Actin

1 2 3 4 1 2 3 4 5 1 2 3 4 5

NC Ad-GFP Ad-HDGF

Pro-collagen I

TGF-ββββ

B

BDL

Control Ad(-) Ad-GFP Ad-HDGF

TG

F- ββ ββ

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

2

4

6

8

10

(n=6) (n=5) (n=5) (n=5)

**

***

BDL

Control Ad(-) Ad-GFP Ad-HDGF

Pro

-co

llag

en αα αα

1(I)

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

2

4

6

8

(n=6) (n=5) (n=5) (n=5)

***

Figure 12. HDGF gene delivery potentiates TGF-β and procollagen α1 protein

expression in BDL-induced fibrotic livers. Mice received intravenous injection of

adenovirus vectors 24 h before liver fibrosis induction. Liver tissues were

collected from mice treated with BDL after 14 days. Total proteins were isolated

for detection of HDGF, TGF-β, and procollagen α1 (I) protein using Western

blotting. (A) Representative images for BDL model. (B) Densitometrical analyses

for respective protein expression in BDL model. All data are presented as mean

induction fold to control ± SE. * , P<0.05; **: P<0.01.

- - 98

A

ββββ-Actin

1 2 3 1 2 3 4 1 2 3 4 5

NC Ad-GFP Ad-HDGF

Pro-collagen I

TGF-ββββ

B

CCl4

Control Ad(-) Ad-GFP Ad-HDGF

TG

F- ββ ββ

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

1

2

3

4

5

6

7

(n=6) (n=5) (n=4) (n=5)

**

**

*

CCl4

Control Ad(-) Ad-GFP Ad-HDGF

Pro

-co

llag

en αα αα

1(I)

exp

ress

ion

(in

du

ctio

n f

old

s to

co

ntr

ol)

0

2

4

6

8

10

(n=6) (n=5) (n=4) (n=5)

**

*

Figure 13. HDGF gene delivery potentiates TGF-β and procollagen α1 protein

expression in CCl4–induced fibrotic livers. Mice received intravenous injection of

adenovirus vectors 24 h before liver fibrosis induction. Liver tissues were

collected from mice treated with CCl4 administration after 35 days. Total proteins

were isolated for detection of HDGF, TGF-β, and procollagen α1 (I) protein using

Western blotting. (A) Representative images for CCl4 model. (B) Densitometrical

analyses for respective protein expression in CCl4 model. All data are presented

as mean induction fold to control ± SE. * , P<0.05; **: P<0.01.

- - 99

Figure 14. HDGF gene delivery deteriorates the collagen deposition in fibrotic livers. Mice received intravenous injection of adenovirus vectors 24 h before liver fibrosis induction. Liver tissues were collected from mice treated with either BDL after

14 days or CCl4 administration after 35 days. Representative microphotographs of Sirus-red staining for immature and

mature collagen fibers. Scale bar = 500 µm.

BDL

CCl4 CCl4+AdGFP CCl4+AdHDGF

BDL+AdGFP BDL+AdHDGF

- 100 -

A B

Control d 7 d 14

% A

rea

of

Co

llag

en D

epo

siti

on

0

1

2

3

4

5

BDL

** *

(n=6) (n=5) (n=5)

% A

rea

of

Co

llag

en D

epo

siti

on

0

2

4

6

8

** ******

Control Ad(-) Ad-GFP Ad-HDGF

BDL

(n=6) (n=5) (n=5)(n=5)

C D

% A

rea

of

Co

llag

en D

epo

siti

on

0

1

2

3

4

Control d 18 d 35

CCl4

***

(n=6) (n=5) (n=4)

% A

rea

of

Co

llag

en D

epo

siti

on

0

1

2

3

4

5

Control Ad(-) Ad-GFP Ad-HDGF

CCl4

** ******

(n=6) (n=5) (n=5)(n=4)

Figure 15. HDGF gene delivery deteriorates the collagen deposition in

fibrotic livers. Morphometrical analysis was performed by randomly selecting

five low-power fields (LPF) in each section to measure the percentage of

collagen deposition area. The results showed that the area of collagen fiber

deposition increased in mouse livers with BDL surgery (A) or CCl4

administration (C). While HDGF gene delivery aggravated intrahepatic

collagen deposition in both BDL (B) and CCl4 (D) models. Data were presented

as mean percentage of total area ± SE. * , P<0.05; **: P<0.01; ***: P<0.001.

- 101 -

A B

TGF-ββββ (ng/ml)0 2 20

HD

GF

gen

e in

du

ctio

n

0

1

2

3

****

Collagen I (-)

TGF-ββββ (ng/ml)

0 2 20

HD

GF

gen

e in

du

ctio

n

0

1

2

3

***

Collagen (+)

Figure 16. TGF-β stimulates HDGF gene expression in cultured hepatocytes

grown on collagen I-coated dishes. Cultured clone-9 hepatocytes were plated

on collagen-uncoated (A) or -coated dishes (B). After overnight serum

starvation, cells were treated with pro-fibrogenic cytokine, TGF-β. After 6h

incubation, total RNA was isolated and subjected to qRT-PCR. The results

showed that TGF-β exhibits a collagen I-dependent HDGF upregulation. Data

are presented as mean induction fold to control ± SD. * , P<0.05, ** , P<0.01.

- 102 -

A

HDGF

Actin

0 2 20 0 2 20 TGF-ββββ (ng/ml)

Collagen (-) Collagen (+)

B

TGF-ββββ (ng/ml)

HD

GF

exp

ress

ion

(in

du

ctio

n f

old

s)

0

1

2

3

** **

* *

0 2 20 0 2 20Collagen (-) Collagen (+)

Figure 17. TGF-β stimulates HDGF protein expression in cultured

hepatocytes grown on collagen I-coated dishes. Cultured clone-9 hepatocytes

were plated on collagen- uncoated (A) or coated dishes (B). After overnight

serum starvation, cells were treated with pro-fibrogenic cytokine, TGF-β. After

24h incubation, lysate protein was collected for Western blotting detection and

followed by densitometrical analysis (C). The results showed that TGF-β

exhibits a collagen I-dependent HDGF upregulation. Data are presented as

mean induction fold to control ± SD. * , P<0.05, ** , P<0.01.

- 103 -

Actin

HDGF

0 50 100 200 0 50 100 200 (M.O.I.)

Ad-GFP Ad-HDGF

Pro-collagen type I

Soluble HDGF

Figure 18. HDGF gene overexpression leads to soluble HDGF release from

cultured hepatocytes. After infection with adenoviral vectors at multiplicity of

infection (MOI) dose of 0, 50, 100, 200 for 48 h, the lysates and supernatants

collected from cultured hepatocytes were subjected to Western blotting

detection. The results showed that HDGF overexpression led to elevation of

pro-collagen I content in and soluble HDGF release from cultured hepatocytes.

- 104 -

A

HDGF conc. (ng/ml)

0 100

TG

F- ββ ββ

gen

e in

du

ctio

n

0

1

2

3

*Collagen (-)

HDGF conc. (ng/ml)

0 100

TG

F- ββ ββ

gen

e in

du

ctio

n

0

1

2

3 Collagen (+)

*

B

HDGF conc. (ng/ml)0 100

TG

F- ββ ββ

(p

g/m

l/105 c

ells

)

0

10

20

30

40

50

*

Collagen (-)

HDGF conc. (ng/ml)

0 100

TG

F- ββ ββ

(p

g/m

l/105 c

ells

)

0

10

20

30

40

50Collagen (+)

*

Figure 19. HDGF overexpression upregulates TGF-β expression in cultured

hepatocytes irrespective of presence of collagen I. (A) qRT-PCR showed that

exogenous HDGF protein enhances TGF-β gene expression. (B) ELISA

measurement revealed that exogenous HDGF protein promotes TGF-β

release from cultured hepatocytes irrespective of presence of collagen I. Data

are presented as mean induction fold to control ± SD. * , P<0.05.

- 105 -

Figure 20. Characterization of α-smooth muscle actin expression in HSC-T6

cells. HSC-T6 cells were seeded on sterile cover glass and cultivated for

overnight till attachment. Cells were fixed by ice-cold methanol and subjected

to imunocytochemical stain. (A) Negative control, isotype-matched mouse IgG

used for primary antibody; (B) positive stain (brownish color). Hematoxylin was

used for counterstaining before mounting. (Bar = 50 µm)

A B

- 106 -

Anti-His Tag Hoechest 33342 Merged

Figure 21. Uptake of exogenous HDGF peptide by cultured HSCs. After 24h

of serum starvation, cultured HSC-T6 cells were treated with recombinant

His-tagged HDGF protein (10 ng/ml) in the presence of 0.5% FBS for 24h. Left,

The HDGF subcellular localization was immunofluorescently visualized using

anti-His antibody. Middle, Hoechst 33342 was used for nuclear counterstaining.

Right, The merged image of both was shown.

- 107 -

HDGF conc. (ng/ml)0 0.1 1 10

Brd

U u

pta

ke(%

of

con

tro

l)

0

80

100

120

140

160

180

200

***

**

Figure 22. HDGF treatment exhibits proliferative effects on cultured HSCs.

After 24h of serum starvation, cultured HSC-T6 cells were treated with HDGF

in the presence of 0.5% FBS for 24h and exposed to BrdU for the last 4h. The

DNA synthesis was determined by BrdU ELISA assay and expressed as mean

percentage of control ± SD. * , P<0.05, ** , P<0.01.

- 108 -

A

B

HDGF conc. (ng/ml)

0 10 30 100

Rel

ativ

e g

ene

exp

ress

ion

(in

du

ctio

n f

old

s)

0

2

4

6

8 αααα-SMA collagen αααα1(I)

**

* *

**

*

Figure 23. HDGF treatment stimulates α-SMA and collagen α1(I) gene

expression in cultured HSCs. Cultured HSC-T6 cells were treated with HDGF

in the presence of 10% FBS for 24h and subjected to qRT-PCR analysis. (A)

Inverted image of DNA electrophoresis after traditional RT-PCR. (B) The

induction folds of gene expression of α-SMA and collagen α1(I) were

measured by using real-time qRT-PCR and normalized to control levels. Data

are presented as mean induction fold to control ± SD. * , P<0.05, ** , P<0.01.

0 10 30 100

αααα-SMA

collagen αααα1(I)

ββββ-actin

+ HDGF (ng/ml)

- 109 -

A

Pro-collagen αααα1 (I)

Actin

αααα-SMA0 0.1 1 10

HDGF (ng/ml)

B C

HDGF conc. (ng/ml)0 0.1 1 10

αα αα-S

MA

exp

ress

ion

(in

du

ctio

n f

old

s)

0

1

2

3

* *

**

HDGF conc. (ng/ml)0 0.1 1 10

Pro

-co

llag

en αα αα

1 (I

) ex

pre

ssio

n(i

nd

uct

ion

fo

lds)

0

1

2**

* *

Figure 24. HDGF treatment stimulates α-SMA and pro-collagen α1(I) protein

expression in cultured HSCs. After 24h of serum starvation, cultured HSC-T6

cells were treated with HDGF in the presence of 0.5% FBS for 24h. The lysates

from cultured HSC-T6 cells treated with HDGF protein were subjected for

Western blotting detection (A). Densitometrical analysis for α-SMA (B) and

procollagen α1 (I) protein expression (C). Data are presented as mean

induction fold to control ± SD. * , P<0.05, ** , P<0.01.

- 110 -

Figure 25. Detection of α-SMA expression in HDGF-treated HSCs by flow

cytometry. Cultured HSC-T6 cells were treated with HDGF in the presence of

10% FBS for 24h. The cells were trypsinized and collected for

immunofluorescent staining followed by flow cytometrical detection. The mean

fluorescent intensity (MFI) and positive rate of a-SMA staining in each group

were analyzed and shown.

Background HDGF (0 ng/ml), % Pos = 97.46 MFI channel # 617.3

HDGF (1 ng/ml), % Pos = 98.72 MFI channel # 654.8

HDGF (10 ng/ml), % Pos = 98.14 MFI channel # 662.0

HDGF (100 ng/ml), % Pos = 97.0 MFI channel # 704.0

HDGF (300 ng/ml), % Pos = 97.53 MFI channel # 688.6

- 111 -

Ad-HDGF Hoechst 33342Ad-GFP

Figure 26. Effects of HDGF gene delivery on HSCs. Left, HSCs were

infected with Ad-GFP for 48h and directly observed under fluorescent

microscope. Middle, Immunofluorescent staining indicates that Ad-HDGF

gene delivery results in HDGF elevation subcellularly localized in both

cytoplasmic and nuclear compartments. Right, Hoechst 33342 was used for

nuclear counterstaining and nucleus is indicated by arrow.

- 112 -

Viral Dose (M.O.I.)

0 12.5 25 50 100 200

Cel

l Pro

lifer

atio

n (

% o

f co

ntr

ol)

0

50

100

150

200Ad-GFP Ad-HDGF

* **** *

Figure 27. Ad-HDGF gene delivery stimulates HSC proliferation. After being

infected with adenoviruses for 24h, cell proliferation was determined by MTS

proliferation assay. Data were presented as mean induction fold to control ±

SD. *: P<0.05, **: P<0.01, compared to corresponding Ad-GFP vehicle

control.

- 113 -

Viral Dose (M.O.I.)

0 12.5 25 50 100 200

Brd

U u

pta

ke (

% o

f co

ntr

ol)

0

20

40

60

80

100

120

140

160

180Ad-GFP Ad-HDGF

*

**

* *

Figure 28. Ad-HDGF gene delivery stimulates DNA synthesis in HSCs. After

being infected with adenoviruses for 24h and exposed with BrdU for 4h, DNA

synthesis was determined by BrdU ELISA assay. Data were presented as

mean induction fold to control ± SD. *: P<0.05, **: P<0.01, compared to

corresponding Ad-GFP vehicle control.

- 114 -

HDGF

Pro-collagen I

Actin

0 1 5 10 25 50 0 1 5 10 25 50 (MOI)Ad-GFP Ad-HDGF

αααα-SMA

Figure 29. Ad-HDGF gene delivery does not affect the expression of

pro-collagen type I and α-SMA proteins in HSCs. After being infected with

adenoviruses at different MOI for 48h, the lysates were subjected to Western

blotting detection.

- 115 -

HDGF ↑↑↑↑

Transdifferentiation

TGF-β β β β ↑↑↑↑

Reciprocal regulation

ECM accumulation

TGF-ββββ

Collagen fiber

HDGF

Hepatocytes

ECMreceptors

ECM ↑↑↑↑

Autocrine

Paracrine

Figure 30. A schematic hypothesis for the pro-fibrogenic role of HDGF.

TGF-β up-regulates HDGF expression in parenchymal hepatocytes during

fibrogenesis. The over-expressed HDGF may be released and autoregulate

TGF-β expression in hepatocytes. Such a unique reciprocal regulation

augments the pro-fibrogenic signaling in fibrotic livers. On the other hand, the

released HDGF peptide also directly exhibits fibroproliferative effects on HSCs

by both expanding HSC population and promoting their transdifferentiation into

myofibroblasts.

- 116 -

A

B

Gel

atin

oly

tic

acti

vity

(arb

itra

ry u

nit

)

0

10000

20000

30000

**

***

#####

###

Figure 31. HDGF not only directly suppresses MMP-2 gelatinolytic activity

but also inhibits the TNF-α-up-regulated MMP-2 activity. 2×105 HSC-T6 cells

were seeded into 6-well culture plate and cultivated for overnight till cell

attachment. Cells were serum-starved 2h before addition of recombinant

TNF-α (20 ng/ml) and HDGF proteins. Then conditioned media were collected

after 24h incubation and subjected to gelatine zymography as described in

‘Materials and Methods’ section.

**: P<0.01, ***: P <0.001, compared to negative group; ##: P <0.01, ###: P

<0.001, compared to TNF-α (+) group.

TNF-αααα ― ― ＋＋＋＋ ＋＋＋＋ ＋＋＋＋ ＋＋＋＋ HDGF ― ＋＋＋＋ ― 1 10 100 (ng/ml)

MMP-2 Pro-MMP-2

118 kD

41 kD

TNF-αααα ― ― ＋＋＋＋ ＋＋＋＋ ＋＋＋＋ ＋＋＋＋ HDGF ― ＋＋＋＋ ― 1 10 100 (ng/ml)

- 117 -

Appendixes

作者簡歷作者簡歷作者簡歷作者簡歷

姓姓姓姓 名名名名：：：：高 英 賢 (Ying-Hsien Kao)

生生生生 日日日日：：：：西元 1967年 1月 15日

出生地出生地出生地出生地：：：：臺灣省屏東縣

主要學歷主要學歷主要學歷主要學歷：：：：

高雄醫學院 醫學技術學系 (1985 ~ 1989)

高雄醫學大學 基礎醫學研究所 (1998 ~ 2001)

國立中山大學 生物科學系 (2004 ~ 2009)

通訊地址通訊地址通訊地址通訊地址：：：：高雄縣鳳山市文教路 68 巷 6號

E-MAI LLLL：：：： danyhkao@gmail.com

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Publication List

A. Peer-Reviewed Articles (#, Co-1st author; *, Corresponding author):

1. Wen-Chuan Wu, Ying-Hsien Kao, Pei-Shin Hu, Jiun-Hwan Chen*.

Geldanamycin, a HSP90 inhibitor, attenuates the hypoxia-induced vascular

endothelial growth factor expression in pigment epithelium cells in vitro. Exp Eye

Res, 85(5):721-731, 2007 (SCI).

2. Ying-Hsien Kao, Shigeru Goto, Bruno Jawan, Toshiaki Nakano, Li-Wen Hsu,

Yu-Chun Lin, Mei-Chun Pan, Chia-Yun Lai, Cheuk-Kuan Sun, Yu-Fan Cheng,

Ming-Hong Tai, Hung-Tu Huang, Chao-Long Chen*. Heat preconditioning

ameliorates hepatocyte viability after cold preservation and rewarming, and

modulates its immunoactivity. Transplant Immunol, 18(3):220-231, 2008 (SCI).

3. Bruno Jawan, Ying-Hsien Kao, Shigeru Goto, Mei-Chun Pan, Yu-Chun Lin,

Li-Wen Hsu, Toshiaki Nakano, Chia-Yun Lai, Cheuk-Kuan Sun, Yu-Fan Cheng,

Ming-Hong Tai, Hock-Liew Eng, Chung-Ren Lin, Chao-Long Chen*. Propofol

pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating

factor production in cultured hepatocytes by suppressing MAPK/ERK activity and

NFκB translocation. Toxicol Appl Pharmacol, 229: 362-373, 2008. (SCI)

4. Ying-Hsien Kao, Bruno Jawan, Shigeru Goto, Chun-Tzu Hung, Yu-Chun Lin,

Toshiaki Nakano, Li-Wen Hsu, Chia-Yun Lai, Ming-Hong Tai, Chao-Long Chen*.

High-mobility group box 1 protein activates hepatic stellate cells in vitro.

Transplant Proc, 40(8): 2704-2705, 2008. (SCI).

5. Yo-Chen Chang, Ying-Hsien Kao#, Dan-Ning Hu, Li-Yu Tsai, Wen-Chuan Wu*.

All trans-retinoic acid remodels extracellular matrix and suppresses

laminin-enhanced contractility of cultured retinal pigment epithelial cells. Exp Eye

Res, 88(5): 900-909, 2009. (SCI)

6. Ying-Hsien Kao, Bruno Jawan, Shigeru Goto, Mei-Chun Pan, Yu-Chun Lin,

Cheuk-Kwan Sun, Li-Wen Hsu, Ming-Hong Tai, Yu-Fan Cheng, Toshiaki Nakano,

Chih-Shien Wang, Chia-Jung Huang, Chao-Long Chen*. Serum factors potentiate

hypoxia-induced hepatotoxicity in vitro through increasing transforming growth

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factor-β1 activation and release. Cytokine, 47(1): 11-22, 2009. (SCI)

7. Ying-Hsien Kao, Bruno Jawan, Cheuk-Kwan Sun, Shigeru Goto, Yu-Chun Lin,

Chun-Tzu Hung, Mei-Chun Pan, Chia-Yun Lai, Li-Wen Hsu, Ming-Hong Tai,

Ching-Chou Tsai, Yu-Fan Cheng, Huoy-Rou Chang, Chao-Long Chen*. High

concentration of magnolol induces hepatotoxicity under serum-reduced conditions.

Phytomedicine (In press), 2009. (SCI)

8. Ying-Hsien Kao, Chao-Long Chen, Bruno Jawan, Yueh-Hua Chung,

Cheuk-Kwan Sun, Shiao-Mei Kuo, Tsung-Hui Hu, Yu-Chun Lin, Hoi-Hung Chan,

Kuang-Hung Cheng, Den-Chyang Wu, Shigeru Goto, Yu-Fan Cheng, David

Chao*, Ming-Hong Tai*. Upregulation of hepatoma-derived growth factor is

involved in murine hepatic fibrogenesis. J Hepatol (Accepted), 2009. (SCI)

B. Oral Presentation in Conferences:

1. Ying-Hsien Kao, Shigeru Goto, Yu-Chun Lin, Bruno Jawan, Hung-Tu Huang,

Chao-Lung Chen. HSP70/Bax ratio of cultured rat hepatocytes is increased by

cold UW solution. 105th Annual Congress of Japan Surgical Society. May 11-13,

2005, Nagoya Congress Center, Nagoya, Japan.

2. Ying Hsien Kao, Bruno Jawan, Shigeru Goto, May-Chung Pan, Yu-Chung Lin,

Cheuk-Kwan Sun, Toshiaki Nakano, Li-Wen Hsu, Chia-Yun Lai, Yu-Fan Cheng,

Chao-Long Chen. Serum factor potentiates the hypoxia-induced hepatocyte cell

death in vitro via TGF-beta activation. The 107th Annual Congress of Japan

Surgical Society. Apr 11-13, 2007, International Conventional Center, Osaka,

Japan. Abstract printed in the Journal of Japan Surgical Society 108: 336, 2007.

3. Ying-Hsien Kao, Bruno Jawan, Shigeru. Goto, Chun-Tzu Hung, Yu-Chun Lin,

Toshiaki. Nakano, Ming-Hong Tai, David Chao, Chao-Long Chen. High-mobility

group box 1 protein activates hepatic stellate cells in vitro. The 10th Congress of

the Asian Society of Transplantation. Dec. 1-4, 2007, Pattaya Exhibition and

Convention Hall, Pattaya, Thailand.

4. Ying-Hsien Kao, Ming-Hong Tai, Yueh-Hua Chung, Bruno Jawan, Shigeru Goto,

Yu-Chun Lin, Toshiaki Nakano, Li-Wen Hsu, Chia-Yun Lai, David Chao, Yu-Fan

Cheng, Chao-Long Chen. Expression profile of hepatoma-derived growth factor in

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fibrotic livers of mice. The 67th Annual Meeting of Taiwan Surgical Association.

Mar. 29-30, 2008, Kaohsiung Veteran General Hospital, Kaohsiung, Taiwan.

5. Ying-Hsien Kao, Ming-Hong Tai, Yueh-Hua Chung, David Chao, Chao-Long

Chen. Hepatoma-derived growth factor up-regulation in mouse fibrotic liver is

involved in hepatic fibrogenesis. The 23rd Joint Annual Conference of Biomedical

Sciences. Mar. 29-30, 2008, National Defense Medical Center, Taipei, Taiwan.