ABSTRACT
BACKGROUND AND OBJECTIVES: Mechanism of regeneration of remnant pancreas after partial pancreatectomy (PX) is still unknown. In this study, effect of siRNA against the collagen specific chaperone, HSP47, which inhibits collagen secretion from activated pancreas stellate cells (aPSCs), and induces their apoptosis, on regeneration of remnant pancreas was determined. METHODS: Pancreatectomy was performed according to established methods. Proliferation of cells was assessed by BrdU incorporation. Immunostaining of HSP47 was employed to identify PSCs. Progenitor cells were identified by SOX9 staining. Acinar cells were immunostained for amylase. Co-culture of acinar cells with aPSCs were carried out in a double chamber with a cell culture insert. siRNA HSP47 encapsulated in vitamin A-coupled liposome (VA-lip siRNA HSP47) was delivered to aPSCs by iv injection. RESULTS: In remnant pancreas of 90% PX rat, new areas of foci were located separately from duodenal areas with normal pancreatic features. After PX, BrdU uptake of acinar cells and islet cells significantly increased, but was suppressed by treatment with VA-lip siRNA HSP47. BrdU uptake by acinar cells was augmented by co-culturing with aPSCs and the augmentation was nullified by siRNA HSP47. BrdU uptake by progenitor cells in foci area was slightly enhanced by the same treatment. New area which exhibited intermediate features between those of duodenal and area of foci, emerged after the treatment. CONCLUSION: aPSCs play a crucial role in regeneration of remnant pancreas, proliferation of acinar and islet cells after PX through the activity of secreted collagen. Characterization of new area emerged by siRNA HSP47 treatment as to its origin is a future task.
Subject(s)
Acinar Cells/cytology , Islets of Langerhans/cytology , Pancreatectomy/rehabilitation , Pancreatic Stellate Cells/cytology , Regeneration/physiology , Stem Cells/cytology , Acinar Cells/metabolism , Animals , Biomarkers/metabolism , Cell Proliferation , Coculture Techniques , Gene Expression , HSP47 Heat-Shock Proteins/antagonists & inhibitors , HSP47 Heat-Shock Proteins/genetics , HSP47 Heat-Shock Proteins/metabolism , Islets of Langerhans/metabolism , Liposomes/administration & dosage , Liposomes/chemistry , Male , Pancreas/drug effects , Pancreas/metabolism , Pancreas/pathology , Pancreas/surgery , Pancreatic Stellate Cells/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Stem Cells/metabolism , Vitamin A/chemistry , Vitamin A/pharmacologyABSTRACT
Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVß1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVß1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.
Subject(s)
Collagen/metabolism , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/metabolism , Matrix Metalloproteinase 14/metabolism , Animals , Apoptosis , Cell Proliferation , Cell Survival , Collagen/antagonists & inhibitors , Collagen/chemistry , HSP47 Heat-Shock Proteins/antagonists & inhibitors , HSP47 Heat-Shock Proteins/genetics , HSP47 Heat-Shock Proteins/metabolism , Hepatic Stellate Cells/drug effects , Humans , I-kappa B Proteins/metabolism , Integrins/antagonists & inhibitors , Integrins/genetics , Integrins/metabolism , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Matrix Metalloproteinase Inhibitors/pharmacology , Mice , Oligopeptides/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Signal Transduction , Tissue Inhibitor of Metalloproteinase-1/antagonists & inhibitors , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolismABSTRACT
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 (3)H-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.
