Mitsuo Tashiro

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.

Role of TGF-β1, Extracellular Matrix, and Matrix Metalloproteinase in the Healing Process of the Pancreas After Induction of Acute Necrotizing Pancreatitis Using Arginine in Rats

Introduction Pancreatic tissues are almost completely restored to normal after an attack of acute pancreatitis, once the cause of the disease is removed. Transforming growth factor (TGF)-&bgr; and extracellular matrix (ECM) are known to play an important role in the process of wound healing in pathologic diseases. Tissue repair is a process regulated by a balance between synthesis and degradation of ECM. Aims To elucidate the role of TGF-&bgr;, ECM, and matrix metalloproteinase (MMP) in the process of regeneration occurring after acute necrotizing pancreatitis. Methodology Acute necrotizing pancreatitis was induced by intraperitoneal injection of 500 mg/100 g body weight of L-arginine in male Wistar rats. Expression of TGF-&bgr;1 and ECM messenger RNA (mRNA) was determined by Northern blot analysis, and that of MMP-1 and MMP-2 mRNA was examined by the reverse transcription polymerase chain reaction (RT-PCR). Immunoreactivity for ECM components, TGF-&bgr;1, and MMP-2 in the pancreas was assessed by using a monoclonal antibody. Results TGF-&bgr;1 mRNA expression reached a peak value on day 2.5, with a decrease on day 3, and reached the control level on day 7. Procollagen types III and IV and fibronectin mRNA reached a peak value on day 2.5, whereas the expression level of procollagen type I mRNA was maximal on day 3, and gradually decreased to control levels by day 7. MMP-2 mRNA was significantly elevated on day 3, and peaked on day 5, whereas MMP-1 mRNA levels did not change throughout the observation period. Immunoreactivity for MMP-2 was observed around disrupted acinar cells and interstitial spaces on day 3, and maximally on day 7. Immunoreactivity for fibronectin was detected around disrupted acinar cells and interstitial spaces. On day 7, it was less than on day 5 around disrupted acinar cells and interstitial spaces, whereas in the regenerated acinar cells, it was undetected. Conclusion Our results show that TGF-&bgr;1 mRNA expression peaked earlier than that of ECM mRNA. Furthermore, increased level of the MMP-2 transcript was followed by disappearance of fibronectin. Our findings suggest that TGF-&bgr;1 plays an important role in ECM production in the early phase of acute pancreatitis, and that MMP-2 is involved in the subsequent healing process.

Green tea polyphenol (–)-epigallocatechin-3-gallate inhibits ethanol-induced activation of pancreatic stellate cells

Background  Activated pancreatic stellate cells (PSCs) play a central role in the pathogenesis of pancreatic fibrogenesis and inflammation. Ethanol, a major cause of chronic pancreatitis, directly induces PSC activation and oxidative stress. Inhibition of PSC activation or stimulation to PSC might be an effective therapeutic strategy for the prevention of pancreatic fibrosis, and (–)‐epigallocatechin‐3‐gallate (EGCG), a major component of green tea extracts, is a potent antioxidant of polyphenols. Therefore, we examined the mechanisms through which ethanol induces oxidative stress on PSCs and evaluated the effect of EGCG on activation and cell functions of ethanol‐stimulated PSCs.

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.

Defects of cholecystokinin (CCK)-A receptor gene expression and CCK-A receptor-mediated biological functions in Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

Abstract: Recent studies in genetically obese and diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats suggest defects of cholecystokinin (CCK)-A receptor gene expression and CCK-A receptor-mediated biological functions such as pancreatic juice, protein, and gastric acid secretion. The present studies were undertaken to further examine CCK-A receptor gene expression and CCK-A receptor-mediated biological functions in the pancreas, stomach, and brain of OLETF rats. Expression of the CCK-A receptor gene could not be detected in the stomach, pancreas and brain by the reverse-transcription polymerase chain reaction (RT-PCR) method and Southern blotting of the PCR products. Southern blot analysis of genomic DNA from OLETF and control Long-Evans Tokushima Otsuka (LETO) rats with CCK-A receptor fragment as a probe revealed different restriction bands. Expression of the CCK-B receptor gene was observed in the stomach, pancreas, and brain in both OLETF and LETO rats by the RT-PCR method, with expression of the CCK-B receptor gene markedly enhanced in OLETF rats compared with that in LETO rats. Consistent with the defect of CCK-A receptor gene expression, CCK-A receptor-mediated biological functions were not observed in these organs. Perfused exocrine and endocrine pancreas of OLETF rats were insensitive to CCK stimulation but not to carbamylcholine stimulation. Basal gastric acid and pepsinogen secretions in OLETF rats were higher than in LETO rats. OLETF rats showed a significantly higher average daily food intake, gained body weight faster, and were heavier than LETO rats. The present study confirmed that OLETF rats have CCK-A receptor gene anomalies and demonstrated deficient CCK-A receptor-mediated biological function in the pancreas, stomach, and brain.

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.

Calcineurin-dependent and calcineurin-independent signal transduction pathways activated as part of pancreatic growth.

Objective: We have recently reported that pancreatic growth driven by cholecystokinin released endogenously by feeding the synthetic trypsin inhibitor camostat requires the Ca2+-activated phosphatase calcineurin. In the present study, we evaluated a number of signal transduction pathways for their activation as part of the growth response and whether their activation was dependent on calcineurin. Methods: Male ICR mice were fed with either chow or chow plus 1 mg/g of camostat. FK506 was administered at 3 mg/kg. After various times from 12 hours to 10 days, pancreatic samples were prepared and assayed for activity of various signal transduction pathway components. Results: Camostat feeding increased the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and phosphorylation of the translation factor eukaryotic initiation factor 4E and activated the mammalian target of rapamycin pathway that leads to phosphorylation of the ribosomal protein S6 and of the eukaryotic initiation factor 4E binding protein but with different time courses. Treatment of mice with the calcineurin inhibitor FK506 totally blocked c-Jun NH2-terminal kinase activation, partially blocked the mammalian target of rapamycin pathway, and had no effect on extracellular signal-regulated kinase activation or the phosphorylation of eukaryotic initiation factor 4E. Conclusions: The pancreatic growth response is accompanied by activation of a number of signaling pathways regulating transcription and translation, some of which are dependent on and some independent of calcineurin.

Ischemic Colitis Associated With Paclitaxel and Carboplatin Chemotherapy

TO THE EDITOR: Combination chemotherapy regimens including paclitaxel have been widely used for standard treatment of many solid tumors. Reported adverse effects on the gastrointestinal tract in paclitaxel-containing chemotherapy regimens are pseudomembranous colitis (1) and gastrointestinal necrosis (2). We describe a case of ischemic colitis (IC) after chemotherapy with paclitaxel and carboplatin. Endoscopic findings of IC are documented. The patient was a 68-yr-old Japanese woman who had been operated on for left upper lobectomy for squamous cell lung cancer. Thirty-seven days after the operation (on day 1), the patient was treated for lymph node metastasis with 135 mg/body of paclitaxel and 240 mg/body of carboplatin at the surgery department of our university hospital. On day 3, she had bloody watery diarrhea five times, with upper abdominal pain, although she had no constipation, fever, nausea, or vomiting. She also had no mucositis or abdominal tenderness. The white blood cell count was increased (15,600/mm), but the C-reactive protein level was normal. On day 5, the patient was consulted at the internal medicine department. Although she had no diarrhea, both white blood cell count and C-reactive protein levels were increased (12,600/mm and 5.3 mg/100 ml, respectively). Colonoscopy showed acute colitis on the right side of the transverse colon. Annular colonic mucosa was hemorrhagic with superficial ulceration (Fig. 1A). It also showed longitudinal ulcerations on the anal side of the hemorrhage with ulceration (Fig. 1B). A pseudomembrane was not detected. A pathological examination of the biopsy specimens showed a mild acute inflammatory infiltration and mild edema with regenerative epithelium, together with a necrotic slough in the colonic mucosa. Stool cultures were normal flora. From these findings, we diagnosed the patient with IC caused by chemotherapy. The patient received total parental nutrition from day 5 to day 14. On day 9, both white blood cell count and C-reactive protein levels returned to normal. On day 14, a barium study showed mild stenosis with ulceration on the right side of the transverse colon indicating healing stage of IC. On day 37, colonoscopy showed a scar on the right side of the transverse colon. Chemotherapy reagents have been implicated in three patterns of necrotizing colitis—pseudomembranous colitis, neutropenic enterocolitis, and IC (3). Pseudomembranous colitis is the common disease after chemotherapy including paclitaxel (1). Several cases of neutropenic enterocolitis after taxane (paclitaxel and docetaxel)-containing chemotherapy have been reported (4–6). Although IC typically develops in people who are otherwise healthy (7), it can also develop after anticancer chemotherapy (3). Ibrahim et al. (4) reported a case of IC as pancolitis after chemotherapy with docetaxel and cyclophosphamide for liver metastasis from breast cancer. Seewaldt et al. (2) also observed and reviewed paclitaxel-associated gastrointestinal necrosis. They postulated that gastrointestinal necrosis is the result of a direct taxane-based effect on the gastrointestinal epithelium (2), although a synergistic interaction between compromised bowel and taxan-induced mitotic arrest is also suggested. Only a single brief report about paclitaxel-associated IC is available. Daniele et al. (8) reported a case of transient mild IC after chemotherapy with paclitaxel and carboplatin for liver metastasis from a neuroendocrine tumor of unknown origin. Colonoscopy is the preferred diagnostic examination for many kinds of colitis because it is more sensitive in diagnosing mucosal abnormalities, and tissue biopsy can be obtained (7). Diarrhea is a common complication of cancer chemotherapy, whereas bloody diarrhea is rare. Therefore, colonoscopy is also important for diagnosis and prompt therapy after chemotherapy with bloody diarrhea (9). Be-

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.

Expression of Survivin after Acute Necrohemorrhagic Pancreatitis in Rats

Introduction Survivin is one of the inhibitors of the apoptosis family and has dual effects: antiapoptotic effect and regulation of the cell cycle. Aim To show involvement of survivin in acute pancreatitis. Methodology Acute necrohemorrhagic pancreatitis was induced in male Wistar rats by intraductal infusion of 4% sodium taurocholate. Results By northern blotting, the survivin mRNA level was significantly increased at 36 hours and peaked at 48 hours after induction of acute pancreatitis. Survivin protein was found in cytoplasm of ductal cells by immunohistochemical analysis at 48–72 hours. It was also observed in nuclei of both acinar and ductal cells as well as infiltrating cells. Apoptotic cells were observed in pancreatic acinar cells. Survivin protein partially colocalized with 5-bromo-2´-deoxyuridine in some nuclei of ductal cells. Conclusions We showed involvement of survivin in acute pancreatitis in rats. Survivin may have some roles in the regulation of pancreatic regeneration and proliferation as well as an antiapoptotic effect after acute pancreatitis.