Taizo Yamaguchi

Inhibition of transforming growth factor beta decreases pancreatic fibrosis and protects the pancreas against chronic injury in mice.

Transforming growth factor-β (TGF-β) is an important cytokine in the fibrogenesis in many organs, including the pancreas. Using an adenoviral vector expressing the entire extracellular domain of type II human TGF-β receptor (AdTβ-ExR), we investigated whether inhibition of TGF-β action is effective against persistent pancreatic fibrosis, and whether it exerts a beneficial effect on the pancreas in the process of chronic injury. To induce chronic pancreatic injury and pancreatic fibrosis, mice were subjected to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 μg/kg body weight cerulein at hourly intervals, per week for 3 consecutive weeks. Mice were infected once with AdTβ-ExR, or with a control adenoviral vector expressing bacterial β-galactosidase (AdLacZ). Pancreatic fibrosis was evaluated by histology and hydroxyproline content. Activation of pancreatic stellate cells (PSCs) was assessed by immunostaining for α-smooth muscle actin. Apoptosis and proliferation of acinar cells were assessed by immunostaining of ssDNA and Ki-67, respectively. Three-week cerulein injection induced pancreatic fibrosis and pancreatic atrophy with proliferation of activated PSCs. In AdTβ-ExR-injected mice, but not AdLacZ-injected mice, pancreatic fibrosis was significantly attenuated. This finding was accompanied by a reduction of activated PSCs. AdTβ-ExR, but not AdLacZ, significantly increased pancreas weight after chronic pancreatic injury. AdTβ-ExR did not change the proportion of proliferating acinar cells, whereas it reduced the number of apoptotic acinar cells. Our results demonstrate that inhibition of TGF-β action not only decreases pancreatic fibrosis but also protects the pancreas against chronic injury by preventing acinar cell apoptosis.

Induction of PDX‐1‐positive cells in the main duct during regeneration after acute necrotizing pancreatitis in rats

Pancreatic regeneration involves two pathways; proliferation and differentiation of pancreatic progenitor cells, which probably exist in pancreatic ductal epithelium, and replication of pre‐existing differentiated acinar, islet, and ductal epithelial cells. During pancreatic development, differentiated cells arise from the ductal progenitor cells expressing the pancreatic/duodenal homeobox‐1 (PDX‐1) homeodomain transcription factor. The aims of this study were to characterize cell proliferation and differentiation during regeneration after acute necrotizing pancreatitis and to evaluate the role of PDX‐1‐positive stem cells. Necrotizing pancreatitis was induced in rats by retrograde intraductal infusion of sodium taurocholate. Cell types were classified into five categories: main, large, and small ductal epithelial cells, tubular complexes and acinar cells. Each category was scored using a 5‐bromo‐2′‐deoxyuridine (BrdU) labelling index (LI) at various time points after induction of pancreatitis. Tissue sections were also immunostained for PDX‐1 to determine the source of pancreatic stem cells. Acinar necrosis was observed at 24 h after induction of pancreatitis and most lobules were filled with tubular complexes on day 5. Subsequently, newly formed acinar cells were observed on day 7, but the lobular architecture returned to normal appearance on day 28. Proliferation started in the main and large ducts at 24 h; marked mitotic activity was evident in small ductal epithelial cells and tubular complexes on day 3, and in acinar cells on day 7. At 24 h after induction of pancreatitis, epithelial cells of the main duct with PDX‐1‐positive nuclei were greatly increased, simultaneously with the peak LI of BrdU. These results suggest that regeneration after necrotizing pancreatitis involves proliferation and differentiation of pancreatic progenitor cells, and that ductal epithelial cells with PDX‐1‐positive nuclei may contribute to the differentiation of pancreatic stem cells in the main duct. Copyright

High glucose activates rat pancreatic stellate cells through protein kinase C and p38 mitogen-activated protein kinase pathway.

Objective: Hyperglycemia is implicated in fibrosis in many organs. Exocrine and endocrine pancreas are closely linked both anatomically and physiologically, and pathological conditions in the exocrine gland can cause impairment of endocrine function and vice versa. Chronic pancreatitis causes pancreatic fibrosis and sometimes results in diabetes mellitus. Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrogenesis. However, the effects of high glucose concentrations on PSC activation have not been fully elucidated. Methods: Cultured PSCs were incubated in the presence of various concentrations of glucose. Pancreatic stellate cell proliferation, &agr;-smooth muscle actin (&agr;-SMA) expression, and collagen production were determined by colorimetric conversion assay, Western blot analysis, and Sirius red dye binding assay, respectively. Results: High glucose concentrations significantly increased PSC proliferation, &agr;-SMA expression, and collagen type I production in PSCs. High glucose concentrations activated protein kinase C (PKC) in PSCs, and PKC inhibitor GF109203X inhibited glucose-stimulated PSC proliferation, &agr;-SMA expression, and collagen secretion. High glucose also activated p38 mitogen-activated protein kinase (MAPK) in PSCs, and p38 MAPK inhibitor SB203580 inhibited glucose-stimulated collagen secretion. Conclusions: Our results indicate that high glucose concentrations stimulate PSC activation via PKC-p38 MAP kinase pathway and suggest that high glucose may aggravate pancreatic fibrosis.

Persistent destruction of the basement membrane of the pancreatic duct contributes to progressive acinar atrophy in rats with experimentally-induced pancreatitis

Background and Aims: The imbalance between synthesis and degradation of extracellular matrix (ECM) proteins is a characteristic feature in chronic pancreatitis. We evaluated the relationship between type IV collagen structure in the basement membrane (BM) and the development of acinar atrophy or the regeneration from acinar injury. Methods: Three different models of pancreatitis were induced in rats by repetitive intraperitoneal injections of 500 mg/100 g body weight of arginine (Arg) or 20 μg/kg body weight of caerulein (Cn) or a single retrograde intraductal infusion of 40 μL/100 g body weight of 3% sodium taurocholate (NaTc). We examined the changes in type IV collagen structure by immunostaining, and the serial changes in the gelatinolytic activity of pro- and active matrix metalloproteinase-2 by zymography in these models of pancreatitis. Results: The pancreas appeared to be histologically normal on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc, whereas 85% to 90% of acinar tissue was replaced by fatty tissue and dilated pancreatic ducts on day 54 after the first intraperitoneal Arg injection. Immunoreactivity for type IV collagen appeared as a discontinuous line along the BM of ducts, vessels, tubular complexes, and acinar cells on day 40 in Arg-induced pancreatitis, whereas it was detected as a continuous line along the BM on day 35 in Cn-induced pancreatitis and on day 42 in NaTc-induced pancreatitits. Gelatinolytic activity of active MMP-2 increased significantly from day 13 to day 40 after the first intraperitoneal Arg injection, whereas it decreased to the baseline level on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc. Conclusions: Our findings indicate that a long-term increase in gelatinolytic activity of active MMP-2 in Arg-induced pancreatitis causes continuous disorganization of type IV collagen in the BM and progressive acinar atrophy, whereas a transient increase in gelatinolytic activity of active MMP-2 is involved in the regeneration of type IV collagen structure in the BM and recovery from acinar injury.

Animal Models of Chronic Pancreatitis

Animal models for CP in rats can be classified into 2 groups: one is noninvasive or nonsurgical models and the other is invasive or surgical models. Pancreatic injury induced by repetitive injections of supramaximal stimulatory dose of caerulein (Cn) or by intraductal infusion of sodium taurocholate (NaTc) recovered within 14 days, whereas that caused by repetitive injection of arginine or by intraductal infusion of oleic acid was persistent. However, the destroyed acinar tissues were replaced by fatty tissues without fibrosis. Transient stasis of pancreatic fluid flow by 0.01% agarose and minimum injury of the pancreatic duct by 0.1% NaTc solution induced progressive pancreatic injury although one alone is insufficient to cause persistent pancreatic injury. However, the damaged tissue was replaced by fatty tissue without fibrosis. Continuous pancreatic ductal hypertension (PDH) caused diffuse interlobular and intralobular fibrosis closely resembling human CP.

Long-term Overexpression of Membrane Type-1 Matrix Metalloproteinase and Matrix Metalloproteinase-2 in Oleic Acid-induced Pancreatitis in Rats

Introduction The matrix metalloproteinase (MMP) plays important roles in extracellular matrix turnover. However, little is known about the roles of MMP-2 and type IV collagen, and the relationship between MMP-2 and membrane type-1 matrix metalloproteinase (MT1-MMP) during progressive destruction of acinar cells in pancreatitis. Aims and Methodology To examine the serial changes in the expression and activity of MMP-2 and expression of MT1-MMP and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in rats after induction of pancreatitis by intraductal infusion of oleic acid, and to determine protein concentrations by Western blot analysis and localization of type IV collagen by immunostaining. Results Gelatinolytic activity of pro-and active MMP-2 and concentrations of MT1-MMP protein, as determined by zymography and Western blot analysis, respectively, increased significantly from 6 hours to day 42 after intraductal infusion of oleic acid. TIMP-2 mRNA expression was significantly higher than that at time 0 throughout the study period, and gelatinolytic activity of active MMP-2 increased from day 3 to day 42. In addition, immunoreactivity for type IV collagen was detected as a discontinuous line along the basement membranes of ducts, vessels, tubular complexes, and acinar cells. Conclusion Our findings indicate that long-term increases of gelatinolytic activity of active MMP-2 cause continuous disorganization of type IV collagen in basement membranes during progressive atrophy of pancreatic gland in oleic acid–induced pancreatitis, and that MT1-MMP and TIMP-2 work as activating factors during proMMP-2 activation. Moreover, basement membranes disorganization in the sustentacula of acinar cells and duct epithelia seems to participate in continuous derangement of acinar cells and duct epithelium.

Oleic acid-induced Pancreatitis alters expression of transforming growth factor-β1 and Extracellular matrix components in rats

Introduction and Aims Extracellular matrix (ECM) components participate in the process of tissue repair and development of fibrosis in the pancreas. We studied the production kinetics of ECM components and transforming growth factor (TGF)–&bgr;1 and identified their production sites in the pancreas following pancreatitis. Methodology Pancreatitis was induced in rats by a single intraductal infusion of oleic acid. Gene expression of TGF-&bgr;s and ECM components was studied by northern blotting. Pancreatic stellate cell activation was assessed by immunostaining for &agr;-smooth muscle actin (&agr;SMA) and desmin. Results Gene expression of TGF-&bgr;s and ECM components was increased in association with pancreatic fibrosis after 1–2 weeks and remained higher than the control levels for the ensuing 12 weeks. Both &agr;SMA and desmin were strongly immunostained around small vessels and faintly stained in mesenchymal cells and tubular complexes at 1 week. The combination of staining for &agr;SMA plus in situ hybridization for procollagen type III mRNA revealed that procollagen type III mRNA was expressed in both &agr;SMA-positive and &agr;SMA-negative cells in the mesenchyma. Conclusions Our findings demonstrate that expression of genes for both TGF-&bgr;s and ECM components was increased and that both &agr;SMA-positive myofibroblasts and mesenchymal cells are the major sources of ECM components after pancreatitis.

Preferential increase of extracellular matrix expression relative to transforming growth factor beta1 in the pancreas during the early stage of acute hemorrhagic pancreatitis in rats.

Objectives: To elucidate the role of transforming growth factor (TGF) β1 and extracellular matrix (ECM) after acute necrotizing pancreatitis, we studied the regulation of TGF-β1 and ECM after induction of pancreatitis. Methods: We examined the serial changes of levels of plasma TGF-β1 by enzyme-linked immunoassay and expression of TGF-β1 and ECM by Northern and Western blot analyses, respectively, in the pancreas after induction of sodium taurocholate-induced acute pancreatitis. Results: Plasma total (active and inactive) TGF-β1 levels at 3 hours after induction of pancreatitis were significantly increased compared with baseline values. The levels of TGF-β1 messenger RNA (mRNA) were unaltered by day 2. Levels of fibronectin mRNA at 3 hours after induction of pancreatitis were already higher than the baseline values. Latency-associated peptide-TGF-β1 showed a peak on day 7. Thus, the expression of ECM mRNA increased earlier than that of TGF-β1 mRNA. Conclusions: These results suggest that the increase in plasma TGF-β1 concentration probably results from the elevated secretion of TGF-β1 from several cells and/or the redistribution of TGF-β1 protein and that the increase in expression of ECM probably is associated with the activation of TGF-β1. It is conceivable that both increased plasma concentration and focal activation of TGF-β1 play an important role in ECM production during the early stage of acute pancreatitis.Abbreviations: AP - acute pancreatitis, ECM - extracellular matrix, EDTA - ethylene diamine tetraacetic acid, FN - fibronectin, NaTc - sodium taurocholate, PBS - phosphate buffered saline, SDS - sodiumdodecyl sulfate, SSC - saline sodium-citrate solution, TGF-β1 - transforming growth factor-β1

Cholecystokinin‐1 receptor protein up‐regulation during pancreatic regeneration after acute haemorrhagic pancreatitis in rats

Background  Cholecystokinin (CCK) plays an important role in regeneration after acute pancreatitis in rats. The present study was aimed to elucidate the role of CCK‐1 receptor (CCK‐1R) in acute pancreatitis. We investigated the serial changes in CCK‐1R mRNA and protein levels and their immunolocalization after acute haemorrhagic pancreatitis induced in male Wistar rats by retrograde intraductal infusion of 4% sodium taurocholate (100 µL 100 g−1 body weight).