woLiver Regeneration Effect of Oncostatin M Following Hepatectomy for the Rat Cirrhotic Liver Model

Background/Aims: Liver resection represents the treatment of choice for hepatocellular carcinoma (HCC) arising in well-compensated cirrhosis. Gene expression of the multifunctional cytokine, Oncostatin M (OSM), stimulates liver regeneration and adenoviral vector expressing OSM (AdOSM) allows a persistent expression of the gene. The aim of this study is to evaluate the benefits of the preoperative injection of AdOSM to the remnant lobes to regenerate the liver. Methods: A 70% partial hepatectomy was performed in dimethylnitrosamine-administrated cirrhotic rats with a preoperative injection of AdOSM, adenoviral vector carrying β-galactosidase (AdLacZ), or phosphate-buffered saline (PBS). The morphologic, histologic, and biochemical changes in the remnant liver and survival rates were then assessed. Results: Portal injection with clamping the portal branches of the resected lobes for 5 min made it possible to effectively transduce the adenoviral vector into the remnant lobes. The ratio of the remnant liver weight/body weight (%) was 2.3 ± 0.5 in the AdOSM group, 1.1 ± 0.3 in the AdLacZ group (p < 0.001), and 1.6 ± 0.4 in the PBS group (p = 0.02). The fibrous ratio (%) was 21.3 ± 4.6 in the AdOSM group and 35.2 ± 4.5 in the AdLacZ group on day 4 after hepatectomy and fibrous status was significantly decreased in the AdOSM group (p = 0.02). Serum hyaluronic acid which is the indicator of liver fibrosis was 215 ± 141 ng/mL in the AdOSM group and 1963 ± 1225 ng/mL in the AdLacZ group (p = 0.03). Conclusions: The OSM gene therapy may increase the possibility of hepatectomy in a cirrhotic liver by improving fibrosis, hepatic function, and hepatocyte regeneration.

• Oncostatin M; Liver Regeneration; Cirrhosis; Anti-Fibrosis

1. Introduction

Although recent demonstrations of satisfactory results of nonsurgical procedures such as percutaneous ethanol injection (PEI), radiofrequency ablation (RFA), and transcatheter arterial chemoembolization (TACE), liver resection still plays an important role in treatment of Hepatocellular carcinoma (HCC) [1]. However, liver failure following hepatectomy sometimes occurs when a liver resection volume exceeds safety limits. This is because the regenerative ability and liver functions are impaired in the patients with cirrhosis. Therefore, one should consider accelerating the remnant liver regeneration to prevent postoperative liver failure [2,3].

Previously administration of hepatocyte growth factor (HGF) was aimed to support liver regeneration for the cirrhotic partial hepatectomy models and showed preferable regeneration effects [4,5]. However, HGF itself has a potential to induce cancer growth or hepatocarcinogenesis and which were the subject to be settled.

On costatin M (OSM), a pleiotropic glycoprotein cytokine that was originally purified from the conditioned media of phorbol ester stimulated U937 (histocytic lymphoma) cells, is related structurally and functionally to the subfamily of neurotrophic cytokines known as the interleukin-6 (IL-6) [6]. The OSM was originally recognized as an inhibitor for tumor cell’s proliferation. However, the OSM presents many other biological activities such as in inflammation, development, and tissue remodeling in the injured liver [7-9].

In the liver, the OSM stimulates the expression of acute phase proteins and tissue inhibitor of metalloproteinase-1 (TIMP-1) [10]. It is well known that TIMP-1 plays an important role in remodeling of extracellular matrix during liver regeneration [11]. Among the IL-6 family cytokines the OSM was the strong inducer of the TIMP-1 [12]. Therefore, administration of the OSM might help to promote the remnant liver regeneration.

In general the cytokines are rapidly turned over, therefore continuous dosage are needed to keep its biological activity. To overcome the problem, several vectors including naked plasmid, liposome and adenoviral vector have been used [13].

The recombinant human adenovirus type 5 is frequently used for in vivo gene therapeutic applications. This virus infects a range of dividing and non dividing cells very efficiently and has an affinity to liver tissue in particular. Although adenovirus-related toxicity remains major concerns [14], it would still be a suitable vector for accelerating liver regeneration.

In this study, we evaluated the benefits of the preoperative injection of adenoviral vector expressing the OSM (AdOSM) to the remnant lobe to accelerate its regeneration following hepatectomy.

2. Material and Methods

2.1. Animals

During all the following experimental procedures, the animals were treated in accordance with Japanese Government Animal Protection and Management Law (No. 105), Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6), the Guidelines for Animal Experiments of Wakayama Medical University, and the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council).

Seven-week-old male, Sprague-Dawley rats that weighed 200 to 250 g were purchased (Kiwa Jikken, Wakayama, Japan) and fed a nutritionally balanced rodent diet and water ad libitum. Liver fibrosis was induced by the intraperitoneal injections of 1% dimethylnitrosamine (DMN) (Nacalai tesque, Kyoto, Japan) dissolved in saline at 1 mL/kg body weight for 3 consecutive days per week for 3 weeks, which showed a pseudolobular formation that was similar to the characteristic pathologic changes found in liver cirrhosis in humans [15].

2.2. Replication-Defective Recombinant Adenoviral Vectors

E1-and E3-region-deleted replication defective adenoviral vectors expressing either human OSM (AdOSM) or β-galactosidase (AdLacZ) under a CA promoter (comprising a cytomegalovirus enhancer and chicken betaactin promoter) were obtained from the RIKEN Bio Resource Center (Tukuba, Japan). These vectors were propagated with 293 cells (Japan Collection of Research Bioresources, Osaka, Japan), which were human embryonic kidney cells transformed by the E1A and E1B genes. The viruses were purified twice with cesium chloride equilibrium centrifugation, then were dialyzed against phosphate buffered saline (PBS) with 10% glycerol, and titrated with 293 cells. Both viral titers of AdLacZ and AdOSM were 5.0 × 10⁸ plaque forming unit (PFU)/mL. To confirm the expression of OSM or LacZ gene, HeLa cells (Japan Collection of Research Bioresources, Osaka, Japan) were cultured with the transduction of AdOSM or AdLacZ, as previously described [4]. Our previous study showed that the injection of high doses of AdLacZ (2 × 10⁹ PFU) led to fetal hepatotoxicity and injection doses ranged from 5 × 10⁸ to 1 × 10⁹ PFU was safe in cirrhotic rats [4]. Therefore, injection dose in this study was also set to be 5 × 10⁸ PFU.

2.3. Experimental Designs

Figure 1 showed schema of this experimental protocol. Four days after the last DMN injection, the rats were placed into 3 groups: rats were injected into the portal vain with either AdLacZ (5.0 × 10⁸ PFU) in 0.5 mL PBS (AdLacZ group; n = 10); AdOSM (5.0 × 10⁸ PFU) in 0.5 mL PBS (AdOSM group; n=10); or only 0.5 mL of PBS (PBS group; n = 10) under ether anesthesia. These rats were performed a 70% partial hepatectomy on day 4 after the portal injections under ether anesthesia according to the method described by our previously report [5]. All the rats in each group were also followed and the living rats were sacrificed on day 4 after hepatectomy to assess the liver regeneration activity.

References

1. K. Uchiyama, M. Ueno, T. Hama, M. Kawai, M. Tani, H. Terasawa, et al., “Recurrence of Primary Hepatocellular Carcinoma after Hepatectomy—Differences Related to Underlying Hepatitis Virus Species,” Hepatogastroenterology, Vol. 52, No. 62, 2005, pp. 591-595.

2. K. Uchiyama, K. Mori, K. Tabuse, M. Ueno, S. Ozawa, T. Nakase, et al., “Assessment of Liver Function for Successful Hepatectomy in Patients with Hepatocellular Carcinoma with Impaired Hepatic Function,” Journal of Hepatobiliary Pancreatic Surgery, Vol. 15, No. 6, 2008, pp. 596-602 doi:10.1007/s00534-007-1326-2

3. M. Ueno, K. Uchiyama, S. Ozawa, S. Hayami, S. Kiriyama and H. Yamaue, “A New Prediction Model of Postoperative Complications after Major Hepatectomy for Hepatocellular Carcinoma,” Digestive Surgery, Vol. 26, No. 5, 2009, pp. 392-329. doi:10.1159/000229973

4. S. Ozawa, K. Uchiyama, M. Nakamori, K. Ueda, M. Iwahashi, H. Ueno, et al., “Combination Gene Therapy of HGF and Truncated Type II TGF-Beta Receptor for Rat Liver Cirrhosis after Partial Hepatectomy,” Surgery, Vol. 139, No. 4, 2006, pp. 563-573. doi:10.1016/j.surg.2005.10.003

5. M. Ueno, K. Uchiyama, M. Nakamori, K. Ueda, M. Iwahashi, S. Ozawa, et al., “Adenoviral Vector Expressing Hepatocyte Growth Factor Promotes Liver Regeneration by Preoperative Injection: The Advantages of Performing Selective Injection to the Remnant Lobe,” Surgery, Vol. 141, No. 4, 2007, pp. 511-519. doi:10.1016/j.surg.2006.10.006

6. T. Hamada, A. Sato, T. Hirano, T. Yamamoto, G. Son, M. Torii, et al., “Oncostatin M Gene Therapy Attenuates Liver Damage Induced by Dimethylnitrosamine in Rats,” American Journal of Pathology, Vol. 171, No. 3, 2007, pp. 872-881. doi:10.2353/ajpath.2007.060972

7. K. Nakamura, H. Nonaka, H. Saito, M. Tanaka and A. Miyajima, “Hepatocyte Proliferation and Tissue Remodeling Is Impaired after Liver Injury in Oncostatin M Receptor Knockout Mice,” Hepatology, Vol. 39, No. 3, 2004, pp. 635-644. doi:10.1002/hep.20086

8. I. Znoyko, N. Sohara, S. S. Spicer, M. Trojanowska and A. Reuben, “Expression of Oncostatin M and Its Receptors in Normal and Cirrhotic Human Liver,” Journal of Hepatology, Vol. 43, No. 5, 2005, pp. 893-900. doi:10.1016/j.jhep.2005.04.020

9. X. J. Dong, H. Zhang, R. L. Pan, L. X. Xiang and J. Z. Shao, “Identification of Cytokines Involved in Hepatic Differentiation of mBM-MSCs under Liver-Injury Conditions,” World Journal of Gastroenterology, Vol. 16, No. 26, 2010, pp. 3267-3278. doi:10.3748/wjg.v16.i26.3267

10. C. D. Richards, M. Shoyab, T. J. Brown and J. Gauldie, “Selective Regulation of Metalloproteinase Inhibitor (TIMP-1) by Oncostatin M in Fibroblasts in Culture,” Journal of Immunology, Vol. 150, No. 12, 1993, pp. 5596-5603.

11. C. Kerr, C. Langdon, F. Graham, J. Gauldie, T. Hara, C. D. Richards, “Adenovirus Vector Expressing Mouse Oncostatin M Induces Acute-Phase Proteins and TIMP-1 Expression in Vivo in Mice,” Journal of Interferon & Cytokine Research, Vol. 19, no. 10, 1999, pp. 1195-1205. doi:10.1089/107999099313145

12. F. M. Botelho, D. R. Edwards and C. D. Richards, “Oncostatin M Stimulates c-Fos to Bind a Transcriptionally Responsive AP-1 Element within the Tissue Inhibitor of Metalloproteinase-1 Promoter,” Journal of Biological Chemistry, Vol. 273, No. 9, 1998, pp. 5211-5218. doi:10.1074/jbc.273.9.5211

13. F. Xue, T. Takahara, Y. Yata, Y. Kuwabara, E. Shinno, K. Nonome, et al., “Hepatocyte Growth Factor Gene Therapy Accelerates Regeneration in Cirrhotic Mouse Livers after Hepatectomy,” Gut, Vol. 52, No. 5, 2003, pp. 694-700. doi:10.1136/gut.52.5.694

14. S. L. Brody and R. G. Crystal, “Adenovirus-Mediated on Vivo Gene Transfer,” Annals of the New York Academy of Sciences, Vol. 716, 1994, pp. 90-101. doi:10.1111/j.1749-6632.1994.tb21705.x

15. D. J. Pritchard and W. H. Butler, “Apoptosis—The Mechanism of Cell Death in Dimethylnitrosamine-Induced Hepatotoxicity,” Journal of Pathology, Vol. 158, No. 3, 1989, pp. 253-260. doi:10.1002/path.1711580314

16. L. Zender and S. Kubicka, “Molecular Pathogenesis and Targeted Therapy of Hepatocellular Carcinoma,” Onkologie, Vol. 31, 2008, pp. 550-555. doi:10.1159/000151586

17. D. E. Cressman, L. E. Greenbaum, R. A. DeAngelis, G. Ciliberto, E. E. Furth, V. Poli, et al., “Liver Failure and Defective Hepatocyte Regeneration in Interleukin-6-Deficient Mice,” Science, Vol. 274, No. 5291, 1996, pp. 1379-1383. doi:10.1126/science.274.5291.1379

18. A. Kamiya, T. Kinoshita, Y. Ito, T. Matsui, Y. Morikawa, K. Nakashima, et al., “Fetal Liver Development Requires a Paracrine Action of Oncostatin M through the gp130 Signal Transducer,” EMBO Journal, Vol. 18, No. 8, 1999, pp. 2127-2136. doi:10.1093/emboj/18.8.2127

19. Y. Ito, T. Matsui, A. Kamiya, T. Kinoshita and A. Miyajima, “Retroviral Gene Transfer of Signaling Molecules into Murine Fetal Hepatocytes Defines Distinct Roles for the STAT3 and Ras Pathways during Hepatic Development,” Hepatology, Vol. 32, No. 6, 2000, pp. 1370-1376. doi:10.1053/jhep.2000.19815

20. T. Kinoshita and A. Miyajima, “Cytokine Regulation of Liver Development,” Biochim Biophys Acta, Vol. 1592, No. 3, 2002, pp. 303-312. doi:10.1016/S0167-4889(02)00323-3

21. T. Yamashita, M. Honda, K. Nio, Y. Nakamoto, T. Yamashita, H. Takamura, et al., “Oncostatin m Renders Epithelial Cell Adhesion Molecule-Positive Liver Cancer Stem Cells Sensitive to 5-Fluorouracil by Inducing Hepatocytic Differentiation,” Cancer Research, Vol. 70, No. 11, 2010, pp. 4687-4697. doi:10.1158/0008-5472.CAN-09-4210