Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Resolution of liver cirrhosis using vitamin A–coupled liposomes to deliver siRNA against a collagen-specific chaperone

Abstract

There are currently no approved antifibrotic therapies for liver cirrhosis. We used vitamin A–coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. Our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin A. Five treatments with the siRNA-bearing vitamin A–coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Rescue was not related to off-target effects or associated with recruitment of innate immunity. Receptor-specific siRNA delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by CCl4 or bile duct ligation. The efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Suppressive effect of siRNAgp46 on gp46 expression and collagen synthesis and RBP receptor dependent uptake of VA-lip-siRNAgp46-FAM by HS cells.
Figure 2: Specific delivery of VA-lip-siRNAgp46 to HS cells in cirrhotic rats.
Figure 3: Effect of i.v. injected VA-lip-siRNAgp46 on cirrhotic rat survival.
Figure 4: Effect of i.v. injection of VA-lip-siRNAgp46 on liver histology of cirrhotic rat.
Figure 5: Effect of i.v. injection of VA-lip-siRNAgp46 on CCl4-treated cirrhotic rats.
Figure 6: Effect of i.v. injection of VA-lip-siRNAgp46 on BDL-induced cirrhotic rats.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Friedman, S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem. 275, 2247–2250 (2000).

    Article  CAS  Google Scholar 

  2. Sauk, J.J., Smith, T., Norris, K. & Ferreira, L. Hsp47 and the translation-translocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen. J. Biol. Chem. 269, 3941–3946 (1994).

    CAS  PubMed  Google Scholar 

  3. Nagata, K. Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol. 16, 379–386 (1998).

    Article  CAS  Google Scholar 

  4. Issa, R. et al. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 126, 1795–1808 (2004).

    Article  CAS  Google Scholar 

  5. Arthur, M.J. Reversibility of liver fibrosis and cirrhosis following treatment for hepatitis C. Gastroenterology 122, 1525–1528 (2002).

    Article  Google Scholar 

  6. Ueki, T. et al. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat. Med. 5, 226–230 (1999).

    Article  CAS  Google Scholar 

  7. Parsons, C.J. et al. Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats. Hepatology 40, 1106–1115 (2004).

    Article  CAS  Google Scholar 

  8. Masuda, H., Fukumoto, M., Hirayoshi, K. & Nagata, K. Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. J. Clin. Invest. 94, 2481–2488 (1994).

    Article  CAS  Google Scholar 

  9. Nagai, N. et al. Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J. Cell Biol. 150, 1499–1506 (2000).

    Article  CAS  Google Scholar 

  10. Bachem, M.G. et al. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 115, 421–432 (1998).

    Article  CAS  Google Scholar 

  11. Fuja, T.J., Probst-Fuja, M.N. & Titze, I.R. Changes in expression of extracellular matrix genes, fibrogenic factors, and actin cytoskeletal organization in retinol treated and untreated vocal fold stellate cells. Matrix Biol. 25, 59–67 (2006).

    Article  CAS  Google Scholar 

  12. Roberts, A.B. et al. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA 83, 4167–4171 (1986).

    Article  CAS  Google Scholar 

  13. Williams, E.J. et al. Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo. Gut 49, 577–583 (2001).

    Article  CAS  Google Scholar 

  14. Murakami, K. et al. Establishment of a new human cell line, LI90, exhibiting characteristics of hepatic Ito (fat-storing) cells. Lab. Invest. 72, 731–739 (1995).

    CAS  PubMed  Google Scholar 

  15. Judge, A.D. et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457–462 (2005).

    Article  CAS  Google Scholar 

  16. Gerling, B., Becker, M., Waldschmidt, J., Rehmann, M. & Schuppan, D. Elevated serum aminoterminal procollagen type-III-peptide parallels collagen accumulation in rats with secondary biliary fibrosis. J. Hepatol. 25, 79–84 (1996).

    Article  CAS  Google Scholar 

  17. Bataller, R. & Brenner, D.A. Liver fibrosis. J. Clin. Invest. 115, 209–218 (2005).

    Article  CAS  Google Scholar 

  18. Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    Article  CAS  Google Scholar 

  19. Marques, J.T. et al. A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat. Biotechnol. 24, 559–565 (2006).

    Article  CAS  Google Scholar 

  20. Kim, D.H. et al. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat. Biotechnol. 22, 321–325 (2004).

    Article  CAS  Google Scholar 

  21. Kim, D.H. et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23, 222–226 (2005).

    Article  CAS  Google Scholar 

  22. Bartlett, D.W. & Davis, M.E. Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res. 34, 322–333 (2006).

    Article  CAS  Google Scholar 

  23. Zimmermann, T.S. et al. RNAi-mediated gene silencing in non-human primates. Nature 441, 111–114 (2006).

    Article  CAS  Google Scholar 

  24. Yano, J. et al. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin. Cancer Res. 10, 7721–7726 (2004).

    Article  CAS  Google Scholar 

  25. Song, E. et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat. Med. 9, 347–351 (2003).

    Article  CAS  Google Scholar 

  26. Flower, D.R. Beyond the superfamily: the lipocalin receptors. Biochim. Biophys. Acta 1482, 327–336 (2000).

    Article  CAS  Google Scholar 

  27. Blomhoff, R., Berg, T. & Norum, K.R. Transfer of retinol from parenchymal to stellate cells in liver is mediated by retinol-binding protein. Proc. Natl. Acad. Sci. USA 85, 3455–3458 (1988).

    Article  CAS  Google Scholar 

  28. Senoo, H. et al. Internalization of retinol-binding protein in parenchymal and stellate cells of rat liver. J. Lipid Res. 31, 1229–1239 (1990).

    CAS  PubMed  Google Scholar 

  29. Senoo, H. et al. Transfer of retinol-binding protein from HepG2 human hepatoma cells to cocultured rat stellate cells. Proc. Natl. Acad. Sci. USA 90, 3616–3620 (1993).

    Article  CAS  Google Scholar 

  30. Fortuna, V.A., Martucci, R.B., Trugo, L.C. & Borojevic, R. Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin. J. Cell. Biochem. 90, 792–805 (2003).

    Article  CAS  Google Scholar 

  31. Noy, N. & Xu, Z.J. Kinetic parameters of the interactions of retinol with lipid bilayers. Biochemistry 29, 3883–3888 (1990).

    Article  CAS  Google Scholar 

  32. Blomhoff, R. & Wake, K. Perisinusoidal stellate cells of the liver: important roles in retinol metabolism and fibrosis. FASEB J. 5, 271–277 (1991).

    Article  CAS  Google Scholar 

  33. Rockey, D.C., Boyles, J.K., Gabbiani, G. & Friedman, S.L. Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J. Submicrosc. Cytol. Pathol. 24, 193–203 (1992).

    CAS  PubMed  Google Scholar 

  34. Osaka, G . et al. Pharmacokinetics, tissue distribution, and expression efficiency of plasmid DNA following intravenous administration of DNA/cationic lipid complexes in mice: Use of a novel radionuclide approach. J. Pharm. Sci. 85, 612–618 (1996).

    Article  CAS  Google Scholar 

  35. Walsh, T.J. et al. Liposomal amphotericin B for empirical therapy in patients with persistent fever and neutropenia. National Institute of Allergy and Infectious Diseases Mycoses Study Group. N. Engl. J. Med. 340, 764–771 (1999).

    Article  CAS  Google Scholar 

  36. Takahashi, N., Tamagawa, K., Shimizu, K., Fukui, T. & Maitani, Y. Effects on M5076-hepatic metastasis of retinoic acid and N-(4-hydroxyphenyl) retinamide, fenretinide entrapped in SG-liposomes. Biol. Pharm. Bull. 26, 1060–1063 (2003).

    Article  CAS  Google Scholar 

  37. Behlke, M.A. Progress towards in vivo use of siRNAs. Mol. Ther. 13, 644–670 (2006).

    Article  CAS  Google Scholar 

  38. Jezequel, A.M. et al. A morphological study of the early stages of hepatic fibrosis induced by low doses of dimethylnitrosamine in the rat. J. Hepatol. 5, 174–181 (1987).

    Article  CAS  Google Scholar 

  39. Farber, J.L. & El-Mofty, S.K. The biochemical pathology of liver cell necrosis. Am. J. Pathol. 81, 237–250 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhou, X. et al. Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis. Liver Int. 24, 492–501 (2004).

    Article  CAS  Google Scholar 

  41. Steffensen, B., Wallon, U.M. & Overall, C.M. Extracellular matrix binding properties of recombinant fibronectin type II-like modules of human 72-kDa gelatinase/type IV collagenase. High affinity binding to native type I collagen but not native type IV collagen. J. Biol. Chem. 270, 11555–11566 (1995).

    Article  CAS  Google Scholar 

  42. Peterkofsky, B. & Diegelmann, R. Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 10, 988–994 (1971).

    Article  CAS  Google Scholar 

  43. Kikuchi, A. et al. Development of novel cationic liposomes for efficient gene transfer into peritoneal disseminated tumor. Hum. Gene Ther. 10, 947–955 (1999).

    Article  CAS  Google Scholar 

  44. Serikawa, T., Suzuki, N., Kikuchi, H., Tanaka, K. & Kitagawa, T. A new cationic liposome for efficient gene delivery with serum into cultured human cells: a quantitative analysis using two independent fluorescent probes. Biochim. Biophys. Acta 1467, 419–430 (2000).

    Article  CAS  Google Scholar 

  45. Schafer, S., Zerbe, O. & Gressner, A.M. The synthesis of proteoglycans in fat-storing cells of rat liver. Hepatology 7, 680–687 (1987).

    Article  CAS  Google Scholar 

  46. Fargnoli, J., Kunisada, T., Fornace, A.J., Jr., Schneider, E.L. & Holbrook, N.J. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc. Natl. Acad. Sci. USA 87, 846–850 (1990).

    Article  CAS  Google Scholar 

  47. Zhang, X. et al. Effects of lipopolysaccharides stimulated Kupffer cells on activation of rat hepatic stellate cells. World J. Gastroenterol. 10, 610–613 (2004).

    Article  CAS  Google Scholar 

  48. Ishak, K. et al. Histological grading and staging of chronic hepatitis. J. Hepatol. 22, 696–699 (1995).

    Article  CAS  Google Scholar 

  49. Jamall, I.S., Finelli, V.N. & Que Hee, S.S. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues. Anal. Biochem. 112, 70–75 (1981).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants-in-aid from Japan Society for the Promotion of Science to Y.S.

Author information

Authors and Affiliations

Authors

Contributions

Y.S., K. Murase and J.K. designed research, performed experiments and wrote the paper. M.K., T.S., Y.K., R.T., K.T., K. Miyanishi, T.M. and T.T. performed experiments. Y.N. designed research, wrote the paper and supervised the whole project. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Yoshiro Niitsu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Tables 1–3, Methods (PDF 1701 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sato, Y., Murase, K., Kato, J. et al. Resolution of liver cirrhosis using vitamin A–coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nat Biotechnol 26, 431–442 (2008). https://doi.org/10.1038/nbt1396

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1396

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing