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

1. 義大醫院 醫學研究部 功能型研究室進度報告 Upregulation of Hepatoma-Derived Growth Factor is Involved in Murine Hepatic Fibrogenesis 實驗室主持人: 高英賢

2. Liver Fibrosis • Definition of fibrosis: as the presence of excessive accumulation of extracellular matrix (ECM) proteins due to new fiber formation. (WHO expert group, 1978) • Liver fibrosis: results from chronic damage to liver in conjunction with ECM deposition, which is a characteristic of most types of chronic liver diseases. • The main causes: chronic HCV infection, alcohol abuse, chronic cholestatic disorders, non-alcoholic steatohepatitis (NASH) • Fibrous scar formed in fibrotic liver distorts the hepatic architecture, and the subsequent development of nodules of regenerating hepatocytes defines cirrhosis, a risk factor for developing hepatocellular cacinoma (HCC).

3. Changes of collagen composition from normal to cirrhotic livers Gressner and Weiskerchen, 2006

4. Role of Hepatic Stellate Cells (HSCs) in Liver Biology • HSCs, also named as Ito cells, fat-storing cells, vitamin A-storing cells, perisinusoidal cells. • Main biological functions: (Sato et al., 2003) 1) Fat and retinoid storage and homeostasis. 2) Remodeling of ECM, by production of both ECM component and matrix metalloproteinases (MMPs). 3) Production of growth factors and cytokines. 4) Contraction and dilation of sinusoidal lumen in response to vasoactive substances. Bataller and Brenner, JCI, 2005

5. Etiologies of Liver Fibrosis Chemical Ethanol, hepatotoxins (CCl4,TTA…) Cholestatic Bile acid… Metabolic Fatty acid, glucose,… Biological HCV, HBV, parasites… Biliary injury Hepatocyte injury Kupffer cell activation Release of free radicals and signaling cytokines HSC activation

6. Hepatoma-Derived Growth Factor (HDGF) • HDGF was purified from the conditioned medium of hepatoma-derived cell line, HuH-7, and has growth-stimulating activity for fibroblasts and hepatoma cells. (Nakamura et al., 1989) • HDGF has a 36% amino acid homology with the high mobility group 1 (HMG-1) protein involved in DNA-binding and postulated to play a role in chromosomal replication and transcription. (Nakamura et al., 1994) • It contains a well-conserved N-terminal amino acid sequence, which is called the HATH(homologous to amino terminus of HDGF) region. (Nakamura et al., 1994) • To date, six members were found in HDGF family: HDGF, HDGF-related proteins (HRP)-1, HRP-2 (Izumoto et al., 1997), HRP-3 (Ikegame et al., 1999), HRP-4 (Dietz et al., 2002), Lens epithelial-cell-derived growth factor (LEDGF) (Ge et al., 1998).

7. Mouse HDGF Protein Family HATH region (1~98 a.a.) (155~170 a.a.) HDGF 237 a.a. in total (211~227 a.a.) HRP-1 283 a.a. in total (321~363 a.a.) HRP-2 669 a.a. in total (136~148 a.a.) HRP-3 203 a.a. in total NLS1 NLS2 Bipartite nuclear localizing signal (NLS)

8. Role of HDGF in Liver Development (Enomoto et al., 2002)

9. Role of HDGF in Hepatocellular Carcinoma (HCC) (Hu et al., 2003)

10. Involvement of HDGF in Tumorigenesis • HCC (Hu et al.; 2003Yoshida et al., 2003 & 2006) • HDGF is a prognostic marker in non-small-cell lung cancer (Ren et al., 2004; Iwasaki et al., 2005). • Involvement in other organ tumors: colorectum (Lepourcelet et al., 2005), stomach (Yamamoto et al., 2006), pancreas (Uyama et al., 2006), esophagus (Yamamoto et al., 2007), and gastrointestinal stromal tumors (Chang et al., 2007).

11. Organogenesis (Morphogenesis) Mitogenesis & Angiogenesis Embryogenesis Tumorigenesis

12. Questions ??? Since liver fibrosis frequently precedes the occurrence of HCC ….. • Whether HDGF is involved in the pathogenesis of liver fibrosis? • Or further, whether HDGF gene knockdown can be an applicable therapeutic strategy to liver fibrosis?

13. Specific Aims • To characterize the expression profile of HDGF during hepatic fibrogenesis and to clarify its pathological significance as compared to fibrotic markers. • To determine the impact of adenovirus-mediated HDGF gene delivery on liver physiological functions. • To determine the impact of HDGF overexpression on the progression of liver fibrosis. • To elucidate the molecular mechanism by which HDGF intervenes in hepatic fibrogenesis.

14. PART I To characterize the expression profile of HDGF during hepatic fibrogenesis and to clarify its pathological significance as compared to fibrotic markers.

15. Materials & Methods • Animals: adult (6~8 w/o) male ICR mice (purchased from National Lab Animal Center, Taipei), performed under the surveillance of the Animal Ethics Committee of Kaohsiung Chang Gung Memorial Hospital. • Biochemical detection for liver function and integrity, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphotase (ALP), g-glutamyltranspeptidase (g-GT), direct bilirubin (D.B.), total bilirubin (T.B.) • RT and quantitative real-time PCR (qRT-PCR, Roche) • Western blotting • Immunohistochemistry (IHC)

16. Animal Models for Liver Fibrosis Induction Day 0 (n=6) Day 7 (n=6) Day 14 (n=6) BDL surgery Day 18 (n=6) Day 35 (n=6) Day 0 1 mL/Kg s.c., twice weekly BDL model CCl4 model

17. Elevated HDGF gene expression in fibrotic livers

18. Elevated HDGF protein levels in fibrotic livers

19. HDGF localization using IHC Normal livers BDL, day 7 CCl4, day 18

20. PART II To determine the impact of adenovirus-mediated HDGF gene delivery on liver physiological functions.

21. Day 0 Day 0 Day 14 Day 14 Animal Treatment Ad-GFP (vehicle control) Ad-HDGF

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

23. Ad-HDGF gene delivery leads to hepatocellular injury and collagen deposition. Data are expressed as mean ±SEM. * indicates p<0.05 as compared to control.

24. PART III To determine the impact of HDGF overexpression on the progression of liver fibrosis.

25. Day 35 Day 0 CCl4 s.c. injection (1 mL/Kg) twice weekly Animal Treatments BDL model CCl4 model Day 0 Day 14 BDL surgery Ad-GFP and Ad-HDGF (109 pfu/mouse) were singly injected through tail vein 24h before BDL surgery or the first CCl4 injection.

26. HDGF gene delivery potentiates TGF-b and procollagen a1 protein expression in fibrotic livers.

27. HDGF gene delivery deteriorates the collagen deposition in fibrotic livers.

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

29. PART IV To elucidate the molecular mechanism by which HDGF intervenes in hepatic fibrogenesis.

30. Mutual regulation between HDGF and TGF-b in cultured hepatocytes

31. Schematic summary

32. Conclusion • We propose that the unique mutual regulation between HDGF and TGF-b expression may augment the profibrogenic signaling during fibrogenesis. • HDGF not only initiates the HSC population expansion at early stages of liver fibrosis, but also enhances the HSC transformation to myofibroblasts by up-regulating a-SMA and collagen protein expression. • Thus, HDGF mechanistically plays an pro-fibrogenic role during hepatic fibrogenesis.

33. Thanks for your attention !