Proliferation Of Pancreatic Stellate Cells Research Articles

Indian hedgehog promotes the migration of rat activated pancreatic stellate cells by increasing membrane type‐1 matrix metalloproteinase on the plasma membrane

Indian hedgehog (Ihh) is a member of hedgehog peptides family that exerts diverse effects on multiple cellular functions. Since Ihh expression is elevated in the pancreas of chronic pancreatitis patients, Ihh has been assumed to participate in the chronic pancreatic injury, especially in pancreatic fibrosis. However, its function in pancreatic fibrosis is still unknown. We thus examined Ihh effects on rat activated pancreatic stellate cells (PSCs) that play a central role in pancreatic fibrosis. Activated PSCs express both patched-1 and smoothened that are essential components of hedgehog receptor system. Ihh did not alter the PSC expression of collagen-1 or alpha-smooth muscle actin, a parameter of PSC transformation, or did not change PSC proliferation. However, Ihh enhanced PSC migration in both chemotactic and chemokinetic manners. Furthermore, Ihh increased the amount of membrane-type 1 matrix metalloproteinase (MT1-MMP) and altered its localization on the plasma membrane, which plays a stimulatory role in cellular migration. In addition, tissue inhibitor of metalloproteinase-2 (TIMP-2) attenuated Ihh-stimulated PSC migration. Since most hedgehog intracellular signals are mediated by Gli-1 transcription factor, we investigated its contribution to Ihh-enhancement of PSC migration. Ihh induced Gli-1 nuclear accumulation in PSCs, indicating that Ihh stimulates Gli-1-dependent signaling pathway in PSCs. Unexpectedly, however, adenovirus-mediated Gli-1 overexpression blocked the Ihh enhancement of both MT1-MMP localization on the plasma membrane and PSC migration. Furthermore, reduction of Gli-1 expression with RNA interference augmented Ihh-stimulated PSC migration. These data indicate that Ihh promotes PSC migration by enhancing MT1-MMP localization on the plasma membrane but is negatively regulated by Gli-1.

Is acute recurrent pancreatitis a chronic disease?

Whether acute recurrent pancreatitis is a chronic disease is still debated and a consensus is not still reached as demonstrated by differences in the classification of acute recurrent pancreatitis. There is major evidence for considering alcoholic pancreatitis as a chronic disease ab initio while chronic pancreatitis lesions detectable in biliary acute recurrent pancreatitis (ARP) seem a casual association. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation, hereditary and obstructive pancreatitis seem an acute disease that progress to chronic pancreatitis, likely as a consequence of the activation and proliferation of pancreatic stellate cells that produce and activate collagen and therefore fibrosis. From the diagnostic point of view, in patients with acute recurrent pancreatitis Endoscopic ultrasound (EUS) seems the more reliable technique for an accurate evaluation and follow-up of some ductal and parenchymal abnormalities suspected for early chronic pancreatitis.

Loss of beta-cells with fibrotic islet destruction in type 2 diabetes mellitus

Recent morphologic analyses of human pancreases strongly suggest that a decreased beta-cell mass is observed from the early stages of diabetes and is caused by accelerated apoptosis of the beta-cells. In this article, we propose that fibrotic islet destruction might be one of the important pathogenic mechanisms of the limited capacity of beta-cell proliferation and accelerated apoptosis in diabetic patients. We have found that pancreatic stellate cells (PSCs) are involved in the progression of islet fibrosis in type 2 diabetes. High concentrations of glucose and insulin in islets contribute to PSC activation and proliferation through angiotensin II type 2 (ATII) signaling pathway, although the exact mechanisms remain to be confirmed. Angiotensin-converting enzyme inhibitors attenuate fibrotic islet destructions and that these have some beneficial effects on glucose tolerance. We suggest that PSCs might play a major role for the fibrotic islet destruction in patients with type 2 diabetes, and suppression of PSCs activation and proliferation might be one of the reasonable target to prevent and delay the progression of the type 2 diabetes mellitus.

Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3

To investigate the role of pancreatic stellate cells (PSCs) and galectin-3 (GAL-3) in the proliferation and infiltration of pancreatic cancer cell line SW1990. Human pancreatic cancer cell line SW1990 and PSCs were cultured in vitro. Supernatant fluid of cultured PSCs and SW1990 cells was collected. Expression of GAL-3 in SW1990 cells and PSCs was detected by ELISA, RT-PCR and Western blotting. Proliferation of cultured PSCs and SW1990 cells was measured by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Infiltration of SW1990 cells was detected by a cell infiltration kit. SW1990 cells expressed GAL-3 and this was up-regulated by the supernatant fluid of cultured PSCs. PSCs did not express GAL-3. SW1990 cells stimulated proliferation of PSCs via GAL-3. GAL-3 antibody inhibited SW1990 cell proliferation, while the supernatant fluid of PSCs stimulated proliferation of SW1990 cells through interaction with GAL-3 protein. The supernatant fluid of PSCs enhanced the invasiveness of SW1990 cells through interaction with GAL-3. GAL-3 and PSCs were involved in the proliferation and infiltration process of pancreatic cancer cells.

High Glucose Activates Rat Pancreatic Stellate Cells Through Protein Kinase C and p38 Mitogen-Activated Protein Kinase Pathway

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. Cultured PSCs were incubated in the presence of various concentrations of glucose. Pancreatic stellate cell proliferation, alpha-smooth muscle actin (alpha-SMA) expression, and collagen production were determined by colorimetric conversion assay, Western blot analysis, and Sirius red dye binding assay, respectively. High glucose concentrations significantly increased PSC proliferation, alpha-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, alpha-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. 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.

Hyperglycemia and hyperinsulinemia have additive effects on activation and proliferation of pancreatic stellate cells: Possible explanation of islet‐specific fibrosis in type 2 diabetes mellitus

Pancreatic islet fibrosis observed in Type 2 diabetes is one of the major factors leading to progressive beta-cell loss and dysfunction. Despite its importance, the mechanism of islet-restricted fibrogenesis associated with pancreatic stellate cell (PSC) activation and proliferation remains to be defined. Therefore, we studied whether the islet-specific environment represented by hyperglycemia and hyperinsulinemia had additive effects on the activation and proliferation of cultured rat PSCs. Cells were stimulated to activate and proliferate with glucose and insulin, either individually or concomitantly. Both stimuli promoted PSC proliferation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation independently, but an additive effect was also demonstrated. Blockade of ERK signaling by the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, suppressed both glucose- and insulin-induced ERK 1/2 phosphorylation and PSC proliferation. Glucose and insulin-induced ERK 1/2 phosphorylation also stimulated connective tissue growth factor gene expression. Thus, hyperglycemia and hyperinsulinemia are two crucial mitogenic factors that activate and proliferate PSCs, and the presence of both states will amplify this response.

Adenovirus-mediated gene transfer of interleukin-4 into pancreatic stellate cells promotes interleukin-10 expression

Pancreatic stellate cells (PSC) are crucially involved in the development of fibrosis, a hallmark of chronic pancreatitis. Therefore, PSC represent an attractive target for the modulation of cellular functions providing the prerequisite for the establishment of novel therapeutic strategies like transfer of genetic material to the cells. Based on recent studies suggesting that the chronic course of pancreatitis is associated with immune deviation towards a Th1 cytokine profile, we have investigated the applicability of primary PSC to an adenovirus-mediated transfer of the cDNA encoding the Th2 cytokine interleukin (IL) 4 and the autocrine-acting effects of IL 4 on the cells in vitro. The trans-duction of primary PSC with a replication-incompetent adenovirus type 5 vector carrying the cDNA encoding rat IL-4 resulted in a distinct expression of the cytokine on mRNA and protein level for two weeks. Similar to recombinant IL 4, effects of the endogenously synthesized cytokine were mediated by the signal transducer and activator of transcription (STAT)6. Interestingly, beside the increase of PSC proliferation, IL 4 transduction was accompanied by an up-regulation in the endogenous expression of the anti-inflammatory cytokine IL 10. In summary, our data suggest that PSC are suitable targets for gene therapy modulating cellular interactions in the pancreas.

Xanthine oxidase‐derived free radicals directly activate rat pancreatic stellate cells

Free radicals are reported to be associated with fibrosis in the pancreas. It is generally accepted that pancreatic stellate cells (PSC) play an important role in pancreatic fibrosis. However, the exact role of free radicals in activation of PSC has not been fully elucidated. In the present study, using a superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DDC) with cultured PSC, we investigated how free radicals act on the activation of PSC. PSC were isolated from male Wister rats. Cultured rat PSC were incubated with DDC for 48 h. Intracellular SOD activity and lipid peroxidation were examined in DDC-treated PSC. Activation of PSC was examined by determining the expression of alpha-smooth muscle actin (alpha-SMA) by immunocytochemistry. The number of PSC using a hemocytometer, type I collagen secretion with ELISA and matrix metalloproteinases (MMP) activities with gelatin zymography were also examined. Secretion of transforming growth factor-beta1 (TGF-beta1) was evaluated by ELISA. The effects of the allopurinol, a xanthine oxidase (XOD) inhibitor, on PSC were also examined. DDC decreased SOD activity and increased lipid peroxidation products in PSC. DDC activated PSC, increasing the number of alpha-SMA positive cells, enhancing secretion of type I collagen and MMP, inhibiting PSC proliferation. Secretion of TGF-beta1, which is known to activate PSC, was increased by DDC treatment. These alterations were prevented by allopurinol. These results suggest that free radicals generated by XOD might directly activate PSC.

Inhibitory effects of interferons on pancreatic stellate cell activation

To analyze and to compare the effects of interferon (IFN)-alpha, IFN-beta, and IFN-gamma on pancreatic stellate cell (PSC) activation in vitro and to elucidate the molecular basis of IFN action. PSCs were isolated from rat's pancreatic tissue, cultured and stimulated with recombinant rat IFNs. Cell proliferation and collagen synthesis were assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA and (3H)-proline into acetic acid-soluble proteins, respectively. Apoptotic cells were determined by FACS analysis (sub-G1 peak method). Exhibition of the myofibroblastic PSC phenotype was monitored by immunoblot analysis of alpha-smooth muscle actin (alpha- SMA) expression. To assess the activation of signal transducer and activator of transcription (STAT), Western blots using phospho-STAT-specific antibodies were performed. In studies on STAT1 function, expression of the protein was inhibited by siRNA. IFN-beta and IFN-gamma but not IFN-alpha significantly diminished PSC proliferation and collagen synthesis. IFN-gamma was the only IFN that clearly inhibited alpha-SMA expression. Under the experimental conditions used, no enhanced rate of apoptotic cell death was observed in response to any IFN treatment. IFN-beta and IFN-gamma induced a strong increase of STAT1 and STAT3 tyrosine phosphorylation, while the effect of IFN-alpha was much weaker. Inhibition of STAT1 expression with siRNA was associated with a significantly reduced growth-inhibitory effect of IFN-gamma. IFN-beta and particularly IFN-gamma display inhibitory effects on PSC activation in vitro and should be tested regarding their in vitro efficiency. Growth inhibition by IFN-gamma action requires STAT1.

High glucose increases extracellular matrix production in pancreatic stellate cells by activating the renin–angiotensin system

Pancreatic stellate cells (PSCs) are involved in pancreatic inflammation and fibrosis. Recent studies have shown that blocking the renin-angiotensin system (RAS) attenuates pancreatic inflammation and fibrosis. However, there are few data about the direct effects of high glucose on extracellular matrix (ECM) protein synthesis and angiotensin II (Ang II) induction in PSCs. PSCs were isolated from male Sprague-Dawley rats and cultured in medium containing 5.5 mM (LG group) or 27 mM D-glucose (HG group). Levels of Ang II and transforming growth factor-beta (TGF-beta) in culture media were measured and Ang II-positive cells were counted. We used real-time polymerase chain reaction (PCR) to detect Ang II receptor expression and Western blot analysis for the expression of ECM proteins such as connective-tissue growth factor (CTGF) and collagen type IV. Cells were also treated with an Ang II-receptor antagonist (candesartan, 10 microM) or angiotensin-converting enzyme (ACE) inhibitor (ramiprilat, 100 nM). Thymidine uptake by PSCs increased fourfold with high glucose treatment. Ang II levels and the proportion of Ang II-positive PSCs were significantly increased after 6 h under high-glucose conditions. TGF-beta concentrations also increased significantly with high glucose. After 72 h, the expression of CTGF and collagen type IV proteins in high-glucose cultures increased significantly and this increase was effectively attenuated by the candesartan or the ramiprilat. All together, high glucose induced PSCs proliferation and ECM protein synthesis, and these effects were attenuated by an Ang II-receptor antagonist. The data suggest that pancreatic inflammation and fibrosis aggravated by hyperglycemia, and Ang II play an important role in this pathogenesis.

Galectin-1 induces chemokine production and proliferation in pancreatic stellate cells

Galectin-1 is a beta-galactoside-binding lectin. Previous studies have shown that galectin-1 was expressed in fibroblasts of chronic pancreatitis and of desmoplastic reaction associated with pancreatic cancer. These fibroblasts are now recognized as activated pancreatic stellate cells (PSCs). Here, we examined the role of galectin-1 in cell functions of PSCs. PSCs were isolated from rat pancreatic tissue and used in their culture-activated phenotype unless otherwise stated. Expression of galectin-1 was assessed by Western blot analysis, RT-PCR, and immunofluorescent staining. The effects of recombinant galectin-1 on chemokine production and proliferation were evaluated. Activation of transcription factors was assessed by EMSA. Activation of MAPKs was examined by Western blot analysis using anti-phosphospecific antibodies. Galectin-1 was strongly expressed in culture-activated but not freshly isolated PSCs. Recombinant galectin-1 increased proliferation and production of monocyte chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant-1. Galectin-1 activated ERK, JNK, activator protein-1, and NF-kappaB, but not p38 MAPK or Akt. Galectin-1 induced proliferation through ERK and chemokine production mainly through the activation of NF-kappaB and in part by JNK and ERK pathways. These effects of galectin-1 were abolished in the presence of thiodigalactosie, an inhibitor of beta-galactoside binding. In conclusion, our results suggest a role of galectin-1 in chemokine production and proliferation through its beta-galactoside binding activity in activated PSCs.

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-β 1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-β 1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-β 1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12- O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-β 1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.

PANCREATIC STELLATE CELL NADPH OXIDASE SYSTEM AND ITS ROLE IN MEDIATING PANCREATIC STELLATE CELL PROLIFERATION BY PLATELET DERIVED GROWTH FACTOR (PDGF)

Activated stellate cells are considered the key mediator in chronic pancreatitis and hepatic cirrhosis. Recently, studies in hepatic stellate cells demonstrated that the reactive oxygen species (ROS) producing enzyme system, NADPH oxidase, regulates proliferation and activation of hepatic stellate cells to produce fibrosis. The aims of this study were to determine whether NADPH oxidase system is present in pancreatic stellate cells, whether growth factors such as PDGF regulate this system and the role of ROS produced by this system in PDGF-mediated pancreatic stellate cell proliferation. We found NADPH oxidase components Nox1, Nox2, Nox4, p22phox, p47phox and p67phox were present at mRNA level in cultured pancreatic stellate cells, Furthermore; Western blot analysis demonstrated the presence of subunits for Nox2, p22phox, p47phox and p67phox in the membrane fraction of pancreatic stellate cells. The NADPH oxidase activity was confined to the membrane fraction of the stellate cells. PDGF treatment significantly increased NADPH oxidase activity in cultured pancreatic stellate cells in a time and dose-dependent manner. PDGF treatment at concentration of 10 ng/ml for 24 hours increased the NADPH oxidase activity by 42% and 260% in serum-enriched (15% FBS) and serum-starved (0.5% FBS) conditions, respectively. These effects were blocked by the NADPH oxidase inhibitor, diphenylene iodonium (DPI). Importantly, PDGF-mediated PSC proliferation was blocked by antioxidant N-acetyl-L-cysteine; ROS scavenger, Tiron and NADPH oxidase inhibitors, DPI and apocynin. Conclusions: NADPH oxidase system is present in pancreatic stellate cells. The activity of NADPH oxidase is modulated by the growth factor, PDGF. NADPH oxidase plays an important role in PDGF-mediated PSC proliferation. These results may provide new insights into mechanisms of pancreatic fibrosis as well as potential molecular target for therapy.

Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α 5β 1 as a Novel CCN2 Receptor

Pancreatic stellate cells (PSCs) are proposed to play a key role in the development of pancreatic fibrosis. The aim of this study was to evaluate the production by rat activated PSCs of the fibrogenic protein, connective tissue growth factor (CCN2), and to determine the effects of CCN2 on PSC function. CCN2 production was evaluated by immunoprecipitation and promoter activity assays. Expression of integrin alpha5beta1 was examined by immunoprecipitation and Western blot. Binding between CCN2 and integrin alpha5beta1 was determined in cell-free systems. CCN2 was assessed for its stimulation of PSC adhesion, migration, proliferation, DNA synthesis, and collagen I synthesis. CCN2 was produced by activated PSCs, and its levels were enhanced by transforming growth factor beta1 treatment. CCN2 promoter activity was stimulated by transforming growth factor beta1, platelet-derived growth factor, alcohol, or acetaldehyde. CCN2 stimulated integrin alpha5beta1-dependent adhesion, migration, and collagen I synthesis in PSCs. Integrin alpha5beta1 production by PSCs was verified by immunoprecipitation, while direct binding between integrin alpha5beta1 and CCN2 was confirmed in cell-free binding assays. Cell surface heparan sulfate proteoglycans functioned as a partner of integrin alpha5beta1 in regulating adhesion of PSCs to CCN2. PSC proliferation and DNA synthesis were enhanced by CCN2. PSCs synthesize CCN2 during activation and after stimulation by profibrogenic molecules. CCN2 regulates PSC function via cell surface integrin alpha5beta1 and heparan sulfate proteoglycan receptors. These data support a role for CCN2 in PSC-mediated fibrogenesis and highlight CCN2 and its receptors as potential novel therapeutic targets.

Pancreatic Stellate Cells (PSCs) express Cyclooxygenase-2 (COX-2) and pancreatic cancer stimulates COX-2 in PSCs

BackgroundCyclooxygenase 2 (COX-2), the inducible form of prostaglandin G/H synthase, is associated with several human cancers including pancreatic adenocarcinoma. Pancreatic stellate cells (PSCs) play a central role in the intense desmoplasia that surrounds pancreatic adenocarcinoma. The present study examined COX-2 expression in PSCs. PSCs isolated from normal rats, were cultured and exposed to conditioned medium (CM) from the human pancreatic cell line, PANC-1.MethodsCOX-2 expression was evaluated by immunostaining and western blotting. Proliferation of PSCs was determined by thymidine incorporation and cell counting.ResultsCOX-2 was found to be constitutively expressed in PSCs, and COX-2 protein was up-regulated by PANC-1 CM. Moreover, the induction of COX-2 by PANC-1 CM was prevented by U0126, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor suggesting that activation of ERK 1/2 is needed for stimulation of COX-2. Finally, NS398, a selective COX-2 inhibitor, reduced the growth of PSCs by PANC-1 CM, indicating that activation of COX-2 is required for cancer stimulated PSC proliferation.ConclusionThe results suggest that COX-2 may play an important role in the regulation of PSC proliferation in response to pancreatic cancer.

Differential and Synergistic Effects of Platelet-derived Growth Factor-BB and Transforming Growth Factor-??1 on Activated Pancreatic Stellate Cells

The cytokines platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta1 are major factors influencing the transformation from the quiescent to the activated phenotype of pancreatic stellate cells (PSC), a process involved in the pathogenesis of chronic pancreatitis. Albeit much effort has been made to study the effects of PDGF and TGF-beta1 on PSCs, their interaction is still unclear, because these cytokines show both differential and synergistic effects as outlined by this study. Culture-activated PSCs of rats were treated with PDGF-BB and TGF-beta1. Subsequent changes of cell proliferation and migration were determined by cell counting, (+)-bromo-2'-deoxyuridine enzyme-linked immunosarbant assay (ELISA), and migration assay. Gene expression, synthesis of proteins, and activation of kinases were further studied by reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, ELISA, and Western blot. PDGF-BB increased PSC proliferation and migration, accompanied by elevated expression of matrix metalloproteinases (MMP)-13 and MMP-3. The mRNA amount of procollagen alpha2(I), alpha-smooth muscle actin (alpha-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and TGF-beta1 was also increased by PDGF-BB. In contrast, PDGF-BB reduced collagen type I in culture medium and synthesis of alpha-SMA. Treatment of PSC with TGF-beta1 decreased proliferation, had no significant effect on migration and MMP expression, but increased expression and synthesis of procollagen alpha2(I) and alpha-SMA. Both cytokines induced phosphorylation of extracellular signal regulated kinase (ERK)-1/2 and p38, but only PDGF-BB activated the protein kinase B signaling pathway. PDGF-BB augments effects of TGF-beta1 on the mRNA level presumably because of up-regulation of TGF-beta1 synthesis and common signaling pathways of the 2 cytokines. However, at the protein level, PDGF-BB impairs typical TGF-beta1 effects such as increased synthesis of collagen (type I) and alpha-SMA. Moreover, PDGF-BB facilitates degradation of extracellular matrix proteins by enhancement of MMP synthesis, but MMP activity was probably limited because of elevated tissue inhibitor of metalloproteinase 1 expression.

Inhibition of lymphocyte apoptosis by pancreatic stellate cells: impact of interleukin-15

There is growing evidence that pancreatic stellate cells (PSCs) produce cytokines and take part in the regulation of inflammatory processes in the pancreas. IL-15 inhibits apoptosis of various cell populations. This study was performed to investigate whether PSCs produce IL-15 and thereby can affect lymphocytes. Primary PSCs were isolated from the rat pancreas using density gradient centrifugation. mRNA expression of IL-15 was demonstrated by RT-PCR, and IL-15 protein was analyzed by immunoblotting. Lymphocytes obtained from rat mesenterial lymph nodes were cocultured with in vitro activated PSCs. Apoptosis has been quantified by the binding of annexin V-FITC with a flow cytometer. Proliferation was monitored using [3H]thymidine incorporation. PSCs express two splice variants of IL-15. The protein was detectable only in cell lysates but not in the cell culture supernatant. Cocultivation of lymphocytes with PSCs and IL-15 inhibited spontaneous lymphocyte apoptosis, and this effect was reduced by an anti-IL-15 antibody. Lymphocytes induced vice versa the proliferation and collagen production of PSCs. The inhibition of spontaneous lymphocyte apoptosis in cocultures with PSCs was at least partially mediated by cell-bound IL-15. This effect and the stimulation of PSCs by lymphocytes may lead to a circulus vitiosus, resulting in the persistence of inflammatory processes and the development of fibrosis during chronic pancreatitis.

Camostat, an oral trypsin inhibitor, reduces pancreatic fibrosis induced by repeated administration of a superoxide dismutase inhibitor in rats

An oral trypsin inhibitor, camostat (CM), has a beneficial effect on chronic pancreatitis, but its mechanism is not yet fully understood. Recently, pancreatic stellate cells (PSC) have been reported to play an essential role in pancreatic fibrosis. An experimental model of pancreatic fibrosis induced by a superoxide dismutase (SOD) inhibitor (diethyldithiocarbamate [DDC]) was developed in rats. Thus, the effect of an oral trypsin inhibitor on pancreatic fibrosis and PSC was investigated. Pancreatic fibrosis was induced in rats using DDC (DDC rats). DDC + CM rats were administered DDC, and subsequently were fed a diet containing CM. Immunohistochemistry of the pancreas was performed with monoclonal anti-alpha-smooth muscle actin (alpha-SMA) antibody and anti-desmin antibody. The DDC rats showed a significant increase in alpha-SMA-positive cells or desmin-positive cells compared with control rats. These significant increases in the fibrotic area improved after treatment with CM. The level of prolyl hydroxylase in the pancreas, which significantly increased as a result of DDC, decreased after treatment with CM. Camostat has a beneficial effect on pancreatic fibrosis induced by the administration of a SOD inhibitor, which inhibits the proliferation and activation of PSC.

Green tea polyphenol epigallocatechin-3-gallate blocks PDGF-induced proliferation and migration of rat pancreatic stellate cells

To clarify the effects of epigallocatechin-3-gallate (EGCG) on the platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of pancreatic stellate cells (PSCs). PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Cell migration was assessed using modified Boyden chambers. Cyclin D1, p21Waf1, and p27Kip1 expression and phosphorylation of PDGF beta-receptor, extracellular signal-regulated kinase, and Akt were examined by Western blotting. Activation of phospha-tidylinositol 3-kinase was examined by kinase assay using phosphatidylinositol as a substrate. Cell cycle was assessed by flow cytometry after staining with propidium iodide. EGCG at non-cytotoxic concentrations inhibited PDGF-induced proliferation and migration. This effect was associated with the inhibition of cell cycle progression beyond the G1 phase, decreased cyclin D1 and increased p27Kip1 expression. EGCG inhibited tyrosine phosphorylation of PDGF beta-receptor and downstream activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways. EGCG inhibited PDGF-BB-induced proliferation and migration of PSCs through the inhibition of PDGF-mediated signaling pathways.

Activation of JAK-STAT pathway is required for platelet-derived growth factor-induced proliferation of pancreatic stellate cells

To clarify the role of Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway in platelet-derived growth factor (PDGF) induced proliferation in activated pancreatic stellate cells (PSCs). PSCs were isolated from rat pancreas tissue, and used in their culture-activated, myofibroblast-like phenotype. STAT-specific binding activity was assessed by electrophoretic mobility shift assay. Activation of Src, JAK2, STAT1, STAT3, and ERK was determined by Western blotting using anti-phospho-specific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. PDGF-BB induced STAT-specific binding activity, and activation of Src, JAK2, STAT1, STAT3, and ERK. Ethanol and acetaldehyde at clinically relevant concentrations decreased basal activation of JAK2 and STAT3. PDGF-induced activation of STAT1 and STAT3 was inhibited by a Src inhibitor PP1 and a JAK2 inhibitor AG490, whereas PDGF-induced activation of ERK was inhibited by PP1, and not by AG490. PDGF-induced proliferation was inhibited by PP1 and AG490 as well as by STAT3 antisense oligonucleotide. PDGF-BB activated JAK2-STAT pathway via Src-dependent mechanism. Activation of JAK2-STAT3 pathway, in addition to ERK, may play a role in PDGF-induced proliferation of PSCs.