Treatment of pancreatic fibrosis with siRNA against a collagen-specific chaperone in vitamin A-coupled liposomes
- Hirotoshi Ishiwatari1,
- Yasushi Sato1,
- Kazuyuki Murase1,
- Akihiro Yoneda2,
- Ryosuke Fujita2,
- Hiroki Nishita2,
- Naoko Kubo Birukawa2,
- Tsuyoshi Hayashi1,
- Tsutomu Sato1,
- Koji Miyanishi1,
- Rishu Takimoto1,
- Masayoshi Kobune1,
- Shigenori Ota3,
- Yasutoshi Kimura3,
- Koichi Hirata3,
- Junji Kato1,
- Yoshiro Niitsu2
- 1Fourth Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan
- 2Department of Molecular Target Exploration, Sapporo Medical University, Sapporo, Japan
- 3First Department of Surgery, Sapporo Medical University, Sapporo, Japan
- Correspondence to Professor Yoshiro Niitsu, Department of Molecular Target Exploration, Sapporo Medical University School of Medicine, South 1, West 17, Chuo-ku, Sapporo 060-8543, Japan;
- Received 27 September 2012
- Accepted 12 October 2012
- Published Online First 20 November 2012
Background and objective Fibrosis associated with chronic pancreatitis is an irreversible lesion that can disrupt pancreatic exocrine and endocrine function. Currently, there are no approved treatments for this disease. We previously showed that siRNA against collagen-specific chaperone protein gp46, encapsulated in vitamin A-coupled liposomes (VA-lip-siRNAgp46), resolved fibrosis in a model of liver cirrhosis. This treatment was investigated for pancreatic fibrosis induced by dibutyltin dichloride (DBTC) and cerulein in rats.
Methods Specific uptake of VA-lip-siRNAgp46, conjugated with 6′-carboxyfluorescein (FAM) by activated pancreatic stellate cells (aPSCs), was analysed by fluorescence activated cell sorting (FACS). Intracellular distribution of VA-lip-siRNAgp46-FAM was examined by fluorescent microscopy. Suppression of gp46 expression by VA-lip-siRNAgp46 was assessed by immunoblotting. Collagen synthesis in aPSCs was assayed by dye-binding. Specific delivery of VA-lip-siRNAgp46 to aPSCs in DBTC rats was verified following intravenous VA-lip-siRNA-FAM and 3H-VA-lip-siRNAgp46. The effect of VA-lip-siRNA on pancreatic histology in DBTC- and cerulein-treated rats was determined by Azan-Mallory staining and hydroxyproline content.
Results FACS analysis revealed specific uptake of VA-lip-siRNAgp46-FAM through the retinol binding protein receptor by aPSCs in vitro. Immunoblotting and collagen assay verified knockdown of gp46 and suppression of collagen secretion, respectively, by aPSCs after transduction of VA-lip-siRNAgp46. Specific delivery of VA-lip-siRNAgp46 to aPSCs in fibrotic areas in DBTC rats was confirmed by fluorescence and radioactivity 24 h after the final injection. 10 systemic VA-lip-siRNAgp46 treatments resolved pancreatic fibrosis, and suppressed tissue hydroxyproline levels in DBTC- and cerulein-treated rats.
Conclusion These data suggest the therapeutic potential of the present approach for reversing pancreatic fibrosis.