for review onlyrdo.psu.ac.th/sjstweb/ar-press/59-dec/25.pdffor review only 2 19 abstract 20 while...

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Abrogation of ALK5 in hepatic stellate cells decreases

hepatic fibrosis and ameliorates liver damage in mice following treatment with thioacetamide

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2016-0456.R1

Manuscript Type: Original Article

Date Submitted by the Author: 14-Dec-2016

Complete List of Authors: Sridurongrit, Somyoth; Mahidol University Faculty of Science, Anatomy

Chen, Ke; Anatomy Kongphat, Wanthita; Graduate program of Toxicology Pudgerd, Arnon; Anatomy Suwannasing, Chanyatip; Anatomy

Keyword: fibrosis, liver injury, TGF-β, ALK5, hepatic stellate cells

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Songklanakarin Journal of Science and Technology SJST-2016-0456.R1 Sridurongrit

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Original article 1

Abrogation of ALK5 in hepatic stellate cells decreases hepatic fibrosis and 2

ameliorates liver damage in mice following treatment with thioacetamide 3

4

Somyoth Sridurongrit1*, Chen Ke

1, Wanthita Kongphat

2, Arnon Pudgerd

1, Chanyatip 5

Suwannasing1 6

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1Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand 8

2Graduate program of Toxicology, Faculty of Science, Mahidol University, Bangkok, 9

Thailand 10

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*Corresponding author: Somyoth Sridurongrit, Ph.D., 1Department of Anatomy, Faculty of 12

Science, Mahidol University, 272 Rama VI road, Rajdhevee, Bangkok 10400, Thailand. 13

Tel: (662) 201-5402; Fax: (662) 201354-7268;Email: [email protected] 14

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Abstract 19

While Transforming growth factor-β (TGF-β) is known to be a key inducer of hepatic 20

stellate cell (hsc) activation during liver fibrosis, it is unclear which TGF-β receptor is 21

required for this hsc-mediated fibrogenesis. Here, we report that abrogation of TGF-β type 22

I receptor ALK5 in hsc led to reduced collagen deposition and decreased number of 23

myofibroblasts in livers of mutant mice lacking ALK5 in hsc (Alk5/GFAP-Cre mice) 24

following thioacetamide (TAA) exposure. The reduced fibrosis was accompanied by 25

decreased expression of hsc activation markers in livers. In addition, Alk5/GFAP-Cre mice 26

exhibited decreased immune cell infiltration and reduced production of inflammatory 27

cytokines. Associated with reduced fibrosis and inflammation, amelioration of liver injury 28

was observed in Alk5/GFAP-Cre mice after TAA treatment. In conclusion, our results 29

indicate that TGF-β signaling via ALK5 in hsc enhanced liver fibrogenesis and 30

inflammation leading to amplification of hepatic injury in mice exposed to TAA. 31

Key words: TGF-β, ALK5, hepatic stellate cells, fibrosis, liver injury 32

1. Introduction 33

Liver fibrogenesis is sustained by a heterogenous population of ECM-producing 34

cells that includes α-smooth muscle actin (α-SMA) positive myofibroblasts (MFs)(Parola 35

et al., 2008). MFs are the major cell type contributing to the accumulation of fibrillar 36

matrix in the fibrous scar (Kisseleva and Brenner, 2011). MFs also promote synthesis and 37

release of growth factors which sustain and perpetuate chronic inflammatory response and 38

neo-angiogenesis in liver (Friedman, 2008b; Forbes and Parola, 2011). In past decades, 39

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various types of studies suggested that MFs could be originated from hepatic stellate cell 40

(hsc) (Friedman, 2008a), portal fibroblasts (Knittel et al., 1999), bone marrow-derived 41

fibrocytes (Asawa et al., 2007), or through an epithelial to mesenchymal transition (EMT) 42

of hepatic epithelium (Zeisberg et al., 2007). Among these cellular sources, MFs were 43

assumed to be mainly derived from hsc (Bataller and Brenner, 2005) since they expressed 44

common fibroblast markers, such as, FSP-1, vimentin, desmin and nestin (Niki et al., 45

1999; Brenner et al., 2012; Strutz et al., 1995; Iwaisako et al., 2012). However, these 46

markers are not only expressed by hsc but also by portal fibroblast, bone marrow-derived 47

cells and epithelial cells (Zhao and Burt, 2007; Eckes et al., 2000; Mendez-Ferrer et al., 48

2010; Osterreicher et al., 2011; Dave and Bayless, 2014). Therefore, it is yet to be further 49

elucidated that the majority of MFs is derived from hsc (Park, 2012). Additional 50

experiments using cell specific gene targeting developed in mice can help us determining 51

the contribution of hsc-derived MFs during the progression of hepatic fibrosis (Forbes and 52

Parola, 2011). 53

Upon liver damage, hsc responds to various liver-damage factors and undergo 54

cellular activation, enabling them to acquire biological function that are pivotal for organ 55

repair (Forbes and Parola, 2011). Among the compelling pathways of hsc activation, TGF-56

β remains a classical family of fibrogenic cytokines that are derived from both paracrine 57

and autocrine sources (Breitkopf et al., 2006; Inagaki and Okazaki, 2007). TGF-β elicits its 58

effects through TGF-β type II (TβR II) and type I (TβR I) cell surface receptors that signal 59

via cytoplasmic serine/theronine kinase domain (Attisano and Wrana, 2002). In hsc, TGF-60

β ligand binding to TβR II leads to recruitment of two subclass of TβR I, ALK5 and 61

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ALK1, that transduces divergent intracellular signaling to regulate gene expression 62

(Goumans et al., 2003). While ALK1 phosphorylation induces the activation of Smad 63

1/5/8 pathway, stimulation of ALK5 results in activation of Smad2/3 cascade (ten Dijke 64

and Hill, 2004). TGF-β signaling has been known to controls collagen gene expression in 65

hsc (Garcia-Trevijano et al., 1999). Although previous studies using mouse models have 66

shown that TGF-β is sufficient to drive hsc activation in vivo (Hellerbrand et al., 1999; 67

Kanzler et al., 1999; Ueberham et al., 2003), it has not been demonstrated which type of 68

TβR I is required in hsc during liver fibrosis. In this study, we show that abrogation of 69

ALK5 signaling in hsc inhibited collagen production in thioacetamide-induced fibrosis 70

mouse model. Furthermore, deficient TGF-β signaling in hsc led to a reduced 71

inflammation and attenuated liver damage induced by toxic stimulus. Our results indicate 72

that ALK5-mediated response in hsc is required for the progression of liver fibrogenesis 73

following liver injury. 74

2. Materials and Methods 75

2.1. Experimental mice 76

To generate mutant mice lacking ALK5 in hsc, Alk5fx/fx

mice (Larsson et al., 2001) were 77

crossed with GFAP-Cre mice, which were also heterozygous for the Alk5fx allele. The 78

resulting homozygotes for Alk5fx alleles, which also carried GFAP-Cre trangene 79

(Alk5fx/fx

/GFAP-Cre+) have ALK5 specifically deleted in hsc (herein termed Alk5/GFAP-80

Cre) whereas littermates with incomplete combination of these alleles served as control. 81

Oligonucleotides for PCR-genetyping of the Alk5fx alleles were previously described 82

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(Dudas et al., 2006). GFAP-Cre mice were obtained from the Jackson Laboratory (Bar 83

Harbor, ME, USA). 84

2.2. Liver injury model 85

All animal studies were conducted with the approval of Animal Ethics Committee on Use 86

and Care of animals at Faculty of Science, Mahidol University, Thailand (Protocol number 87

257). Liver injury was induced by intraperitoneal thioacetamide (TAA) injection three 88

times a week for 12 weeks (100 µg/g body weight for one week, 150 µg/g body weight for 89

one week and 200 µg/g body weight for ten weeks). 90

2.3. Histological analyses and immunostaining 91

For histology, liver tissue were fixed with Bouin solution (Bio-Optica) for 24 hours, 92

dehydrated and embedded in paraffin. Liver sections (6 µm) were stained with 93

hematoxylin and Eosin. To analyze liver fibrosis, sections were stained with Sirius Red 94

(saturated picric acid containing 0.1% direct red and 0.1% Fast green FCF, Sigma). Five 95

Sirius Red-stained slides per mouse were generated, with six 10x pictures taken randomly 96

per slide for a total of 30 images per mouse. The images were analyzed for Sirius Red-97

positive area using ImageJ program. Five mice per experimental group were used for 98

collagen quantification. For immunohistochemistry, sections were stained with antibodies 99

for α-SMA (M085, Dako), myeloperoxidase(PA5-16672, Thermo Fisher Sci.), CD3( 100

A0452, Dako) and cleaved caspase-3 (#9664, Cell Signaling Tech.). 101

102

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2.4. Real-time PCR analyses 103

RNAs from livers were extracted using RNeasy mini kit (Qiagen). QuantiNova RT kit 104

(Qiagen) was used to synthesize cDNA from 1µg of RNA. Quantitative RT-PCRs were 105

carried out with Applied Biosystems real-time PCR 7500 using iTaq Universal SYBR 106

green supermix (Bio-Rad Lab.). Data were represented as the relative expression of gene 107

after normalizing to β-actin. All primer pairs have been previously described (Basciani et 108

al., 2004; De Filippo et al., 2008; Bopp et al., 2013; Yang et al., 2013; Stewart et al., 109

2014). 110

2.6. Statistical analysis 111

All experiments were performed with at five animals per experimental group and 112

representative data were presented as mean + SD. Comparisons were performed by 113

student’s t-test and differences were consider significant if P < 0.05. 114

3. Results 115

3.1. Thioacetamide-induced fibrosis is reduced in Alk5/GFAP-Cre livers 116

Since TGF-β was known to promote hsc activation giving rise to collagen-117

producing myofibroblasts (Hellerbrand et al., 1999), we hypothesized that abrogation of 118

ALK5 in hsc would attenuate liver fibrogenesis. To test this idea, we analyzed the extent of 119

liver fibrosis in Sirius red-stained liver sections obtained from TAA-treated control and 120

TAA-treated Alk5/GFAP-Cre mice (Fig.1). While control livers displayed intense Sirius 121

red-positive staining in fibrotic lesions that connected among perivenular areas to form 122

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pseudolobules (Fig.1A and B), fibrotic lesions in Alk5/GFAP-Cre livers were weakly 123

stained by Sirius Red staining (Fig.1C and D). Morphometric analysis of Sirius red-stained 124

sections showed that collagen deposits were significantly reduced in livers of TAA-treated 125

Alk5/GFAP-Cre mice compared to those of TAA-treated control mice (Fig.2). To 126

determine whether an amount of myofibroblasts coincides with decreased fibrosis in 127

Alk5/GFAP-Cre mice, we stained liver sections with α-SMA antibody. Our results showed 128

that a number of α-SMA-positive cells were observed in the perivenular areas and fibrotic 129

lesions of TAA-treated control livers (Fig.3A) whereas fewer α-SMA-positive cells were 130

seen in livers of Alk5/GFAP-Cre mice (Fig.3B). Consistent with this finding, quantitative 131

RT-PCR (qRT-PCR) showed that mRNA expression of α-SMA was greater in livers of 132

TAA-treated control compared to those of TAA-treated Alk5/GFAP-Cre mice (Fig.3C). 133

Additionally, we assessed whether hepatic expression of key hsc activation markers 134

(Czochra et al., 2006; Thieringer et al., 2008; Mann and Marra, 2010) was reduced in 135

Alk5/GFAP-Cre mice following TAA treatment. Fig.4 shows that PDGF-A, PDGF-B and 136

TGF-β mRNA levels were decreased in livers of TAA-treated Alk5/GFAP-Cre mice 137

compared to those of TAA-treated control mice. 138

Although previous in vitro studies have shown that both type I receptors ALK5 and 139

ALK1 are used by hsc (Khimji et al., 2008), a requirement of these receptors in hsc during 140

TGF-β–mediated liver fibrosis has not been elucidated. Here, we report on a new hsc-141

specific, knockout mouse model that clarify a role of TGF-β signaling via ALK5 in hsc 142

during liver fibrosis progression. The results from our work demonstrated that genetic 143

ablation of ALK5 in hsc led to a decrease in collagen gene expression in liver of mice 144

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exposed to TAA. These data suggested that ALK5 signaling is necessary for hsc-dependent 145

collagen synthesis during liver fibrogenesis. We also found that loss of ALK5 in hsc 146

inhibited TAA-induced myofibroblast production in livers. These findings delineated an 147

essential role of ALK5 during the transformation of quiescent hsc to activated hsc in vivo. 148

3.2. Reduced hepatic inflammatory response in Alk5/GFAP-Cre mice after TAA 149

exposure 150

Because activated hscs/myofibroblasts play important roles not only in promoting 151

fibrosis but also influencing liver inflammation (Novo et al., 2009), it is possible that 152

reduced number of myofibroblasts in Alk5/GFAP-Cre livers may lead to attenuation of 153

inflammatory response to TAA-induced injury. To investigate this possibility, 154

myeloperoxidase and CD3 immunostaining was performed to assess liver infiltration of 155

neutrophils and T lymphocytes, respectively (Fig.5). Immunohistochemistry for 156

myeloperoxidase showed a higher number of myeloperoxidase-positive cells in sinusoids 157

of control livers (Fig.5A and B) than those in Alk5/GFAP-Cre livers (Fig.5E and F). 158

Similar results were obtained from CD3 immunostaining. While a plenty of CD3-positive 159

cells were concentrated around perivascular areas of TAA-treated control livers (Fig.5C 160

and D), fewer CD3-positive cells were found in livers of Alk5/GFAP-Cre mice (Fig.5G 161

and H). In accordance with the reduced immune cell infiltration, hepatic expression of 162

inflammatory cytokines: TNF-α, IL-6, MIP and MCP were decreased in TAA-treated 163

Alk5/GFAP-Cre mice compared to those of TAA-treated control mice (Fig.6). Since many 164

inflammatory cytokines were known to be regulated of TGF-β signaling (Chen et al., 2008; 165

Park et al., 2003; Zhang et al., 2009) and hscs were shown to modulate immunological 166

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response in livers by secreting several chemokines (Friedman, 2008a; Hernandez-Gea and 167

Friedman, 2011), it is tempting to speculate that reduced immune infiltration in mutant 168

livers may be attributed to reduced expression of inflammatory factors by hscs. Further 169

studies are needed to clarify whether TGF-β/ALK5 signaling in hscs could directly 170

mediate liver inflammation via the production of these cytokines and chemokines. 171

3.3. Alk5/GFAP-Cre mice showed amelioration of TAA-induced liver damage 172

Having found a decreased liver fibrosis and inflammation in TAA-treated 173

Alk5/GFAP-Cre mice, we speculated that inhibition of TGF-β/ALK5 signaling in hsc is 174

associated with reduced liver injury. To investigate an extent of liver injury, we analyzed 175

H&E-stained liver sections of TAA-treated control (Fig.7A-C) and TAA-treated 176

Alk5/GFAP-Cre mice (Fig.7D-F). While apoptotic bodies were often found in the 177

perivenular (Fig.7B) and periportal areas (Fig.7C) in livers of TAA-treated control mice, 178

this degenerative change was rarely seen in TAA-treated Alk5/GFAP-Cre livers (Fig.7E 179

and F). In line with this finding, immunohistochemistry for cleaved caspase 3 showed a 180

lower number of cleaved caspase 3-positive cells in livers of TAA-treated Alk5/GFAP-Cre 181

mice than that of TAA-treated control mice (Fig.8). Additionally, serum Asparate 182

transaminase (AST) and Alanine Transaminase (ALT) valves were significantly reduced in 183

TAA-treated Alk5/GFAP-Cre mice when compared to those of TAA-treated control mice 184

(Fig.9). 185

It is becoming clearer that hsc mediates amplification of fibrosis–associated liver 186

damage. Works by Puche et al. (Puche et al., 2013) and Stewart et al. (Stewart et al., 2014) 187

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have demonstrated that hsc contributes to hepatocyte death and exacerbate liver injury 188

caused by a variety of toxic stimuli. These data from our studies further suggested that the 189

pivotal functions of hsc are mediated by TGF-β/ALK5 signaling. We have shown that 190

fibrosis and liver damage was reduced because TGF-β signaling in hsc was blocked by 191

genetic ablation of ALK5. Although the precise mechanism of how TGF-β mediates hsc-192

dependent liver damage remains to be elucidated, our findings point to ALK5 signaling as 193

a fundamental pathway directing liver damage. Therefore, an abrogation of ALK5 194

signaling in hsc by which amplify chronic liver injury has potential therapeutic benefit for 195

fibrosis-associated liver diseases. 196

4. Conclusion 197

In summary, we showed that Tgf-β type I receptor ALK5 was essential for hsc-dependent 198

myofibroblast production during liver fibrosis. In addition, abrogation of ALK5 leaded to 199

attenuation of liver inflammation and hepatic injury following TAA exposure. While 200

further studies are still needed to investigate the mechanism in which hsc-specific TGF-β 201

signaling influences inflammatory response and hepatocellular damage, our findings 202

supported the beneficial effect of hsc-specific ALK5 inhibition as promising treatment for 203

chronic liver injury. 204

5. Acknowledgements 205

This study was supported by TRF grant number TRG5780070 (From Thailand Research 206

Fund in collaboration with Mahidol University), and by funds for new principal 207

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investigators from Mahidol University. This research is also partially supported by Central 208

Instrument Facility (CIF), Faculty of Science, Mahidol University. 209

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liver. Proceedings of National Acadamy of Science U S A. 108, 308-313. 310

Park, J.I., Lee, M.G., Cho, K., Park, B.J., Chae, K.S., Byun, D.S., Ryu, B.K., Park, Y.K. 311

and Chi, S.G. 2003. Transforming growth factor-beta1 activates interleukin-6 312

expression in prostate cancer cells through the synergistic collaboration of the 313

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4332. 315

Park, S.M. 2012. The crucial role of cholangiocytes in cholangiopathies. Gut and liver. 6, 316

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H.F., Jr. and Friedman, S.L. 2013. A novel murine model to deplete hepatic stellate 322

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Lentsch, A.B. and Gandhi, C.R. 2014. A novel mouse model of depletion of stellate 326

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Spontaneous hepatic fibrosis in transgenic mice overexpressing PDGF-A. Gene. 336

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Figure 1. Representative images of Sirius red/fast green-stained liver sections obtained 11

from TAA-treated control and TAA-treated Alk5/GFAP-Cre mice. In TAA-treated control 12

livers, Sirius red-stained collagen fibers were seen in fibrotic lesions that separated liver 13

parenchyma into pseudolobules (A and B). While developing fibrous bridgings were seen 14

in livers of TAA-treated Alk5/GFAP-Cre mice, Sirius red-positivity was greatly reduced in 15

fibrotic lesions and perivenular areas when compared to those of TAA-treated control mice 16

(C and D).FL, fibrotic lesions; VS, vascular space; *, pseudolobule. 17

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Figure 2. Morphometric analysis of Sirius red-stained sections of TAA-treated control and 29

TAA-treated Alk5/GFAP-Cre mice. Data are mean + SD of n = 5 per experimental group. 30

*P < 0.05, significantly different compared with saline-treated control group. There was 31

significant differences compared with TAA-treated control group (**P < 0.05). 32

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Figure 3. Representative images of immunohistochemical staining of α-SMA on liver 45

tissue sections. After TAA exposure, control livers showed intense α-SMA-46

immunoreactivity (red) around perivenular areas as well as in fibrotic lesions (A). In livers 47

of TAA-treated Alk5/GFAP-Cre mice, less α-SMA-positivity was detected around portal 48

and peritotal areas (B). Quantitative RT-PCR (qRT-PCR) analysis showed a lower hepatic 49

α-SMA mRNA expression in TAA-treated Alk5/GFAP-Cre mice, compared to those of 50

TAA-treated control mice (C). Data were normalized to β-actin mRNA level. *, 51

significantly different from TAA-treated control at P <0.05 (student’s t-test). 52

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Figure 4. Relative mRNA expression of hsc activation marker: PDGF-A, PDGF-B and 63

Tgf-β. Expression levels were normalized to β-actin mRNA level. Five mice per 64

experimental group were used for the analysis. *, significantly different from TAA-treated 65

control at P <0.05 (student’s t-test). 66

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Figure 5. Immunohistochemistry for neutrophil: myeoloperoxidase and for T lymphocyte: 79

CD3 in liver of TAA-treated mice. While a number of myeoloperoxidase-postive cells 80

were evenly distributed throughout liver parenchyma of TAA-treated control mice (A and 81

B), myeoloperoxidase-postivity was hardly detected in TAA-treated Alk5/GFAP-Cre livers 82

(E and F). Immunostaining for CD3 showed that CD3-positive cells were concentrated 83

around fibrotic lesions and perivascular areas in control livers (C and D) whereas CD3-84

immunoreactivity was less seen in Alk5/GFAP-Cre livers (G and H). 85

Immunohistochemistry with NovaRED substrate, counterstained with hematoxylin. 86

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Figure 6. Hepatic mRNA expression of inflammatory cytokines in TAA-treated mice. 97

qRT-PCR analysis of TNF-α, IL-6, MIP and MCP expression was performed by cDNA 98

obtained from livers TAA-treated mice. Expression levels were normalized to β-actin 99

mRNA level. *, significantly different from TAA-treated control at P <0.05 (student’s t-100

test). Five mice per experimental group were used for the analysis. 101

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Figure 7. Histological changes of TAA-induced injury in control (A-C) and Alk5/GFAP-116

Cre mice (D-F). Apoptotic bodies were frequently seen around central veins (B, arrow) 117

and in periportal areas (C, arrow) in TAA-treated control livers.This degenerative change 118

was less seen in livers of Alk5/GFAP-Cre mice (D-F). Liver samples were stained for 119

hematoxylin and eosin. CV, central vein. 120

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Figure 8. Immunohistochemistry for cleaved caspase 3 in livers of TAA-treated mice. 134

Cleaved caspase3-postive cells (brown) were often found in perivenular areas of TAA-135

treated control mice (A and B). Decreased number of cleaved caspase3-postive cell was 136

observed in livers of TAA-treated Alk5/GFAP-Cre mice (C and D). 137

Immunohistochemistry with NovaRED substrate, counterstained with hematoxylin. VS, 138

vascular space. 139

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Figure 9. Analysis of serum liver enzyme level. The level of serum Asparate transaminase 152

(AST) and Alanine transaminase (ALT) in TAA-treated Alk5/GFAP-Cre mice was lower 153

than that of TAA-treated control mice. Five mice from each experimental group were used 154

for the analysis. There was significant differences compared with TAA-treated control 155

group (*P < 0.05). 156

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Supplementary figure 1. mRNA expression analysis of Alk5 in hepatic stellate cell isolated from

control and Alk5/GFAP-Cre livers. Successful gene recombination of floxed Alk5 gene was

evaluated by RT-PCR using primers specific for sequences in exon 2 and 4. While control

sample displayed a full-range Alk5 mRNA (399-bp fragment), Alk5/GFAP-Cre sample showed

a 156-bp PCR products representing the Alk5 mRNA lacking exon3.

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