Proliferation Of Pancreatic Stellate Cells Research Articles

PSCs and GLP-1R: occurrence in normal pancreas, acute/chronic pancreatitis and effect of their activation by a GLP-1R agonist

There is increasing concern about the development of pancreatitis in patients with diabetes mellitus who received long-term glucagon-like peptide-1 (GLP-1) analog treatment. Its pathogenesis is unknown. The effects of GLP-1 agonists on pancreatic endocrine cells are well studied; however, there is little information on effects on other pancreatic tissues that might be involved in inflammatory processes. Pancreatic stellate cells (PSCs) can have an important role in pancreatitis, secreting various inflammatory cytokines/chemokines, as well as collagen. In this study, we investigated GLP-1R occurrence in normal pancreas, acute pancreatitis (AP)/chronic pancreatitis (CP), and the effects of GLP-1 analog on normal PSCs, their ability to stimulate inflammatory mediator secretion or proliferation. GLP-1 receptor (GLP-1R) expression/localization in normal pancreas and pancreatitis (AP/CP) tissues were evaluated with histological/immunohistochemical analysis. PSCs were isolated from male Wistar rats. GLP-1R expression and effects of GLP-1 analog on activated PSCs was examined with real-time PCR, MTS assays and western blotting. In normal pancreas, pancreatic β cells expressed GLP-1R, with only low expression in acinar cells, whereas in AP or CP, acinar cells, ductal cells and activated PSCs expressed GLP-1R. With activation of normal PSCs, GLP-1R is markedly increased, as is multiple other incretin-related receptors. The GLP-1 analog, liraglutide, did not induce inflammatory genes expression in activated PSCs, but induced proliferation. Liraglutide activated multiple signaling cascades in PSCs, and the extracellular signal-regulated kinase pathway mediated the PSCs proliferation. GLP-1Rs are expressed in normal pancreas and there is marked enhanced expression in AP/CP. GLP-1-agonist induced cell proliferation of activated PSCs without increasing release of inflammatory mediators. These results suggest chronic treatment with GLP-1R agonists could lead to proliferation/chronic activation of PSCs, which may lead to important effects in the pancreas.

INS-1 cells inhibit the production of extracellular matrix from pancreatic stellate cells

In type 2 diabetes mellitus, pancreatic stellate cells (PSCs) are present within and surrounding pancreatic islets and may cause progressive fibrosis and deterioration of pancreatic beta cell function. However, it is unknown whether pancreatic beta cells influence the biological behavior of PSCs. In the present study, we examined the impact of pancreatic beta cells on the proliferation, migration and extracellular matrix (ECM) production of PSCs. PSCs were treated with conditioned media from INS-1 cells (supernatant, SN). Although the proliferation of PSCs incubated with INS-1-SN was increased compared to control, INS-1-SN treatment induced matrix metalloproteinase-2 activity and reduced the production of ECM and TGF-β1. In addition, PSCs treated with INS-1-SN reduced the secretion of cytokines that are known to mediate pancreatic beta cell death, such as FADD, Fas, IFN-γ, IL-1, TNF-α, and TRAIL. Our findings suggest that pancreatic beta cells may ameliorate islet fibrosis and the progression of islet dysfunction.

Extracellular matrix composition significantly influences pancreatic stellate cell gene expression pattern: role of transgelin in PSC function

Activated pancreatic stellate cells (PSCs) are responsible for the fibrotic matrix of chronic pancreatitis and pancreatic cancer. In vitro protocols examining PSC biology have usually involved PSCs cultured on plastic, a nonphysiological surface. However, PSCs cultured on physiological matrices, e.g., Matrigel (normal basement membrane) and collagen (fibrotic pancreas), may have distinctly different behaviors compared with cells cultured on plastic. Therefore, we aimed to 1) compare PSC gene expression after culture on plastic, Matrigel, and collagen I; 2) validate the gene array data for transgelin, the most highly dysregulated gene in PSCs grown on activating vs. nonactivating matrices, at mRNA and protein levels; 3) examine the role of transgelin in PSC function; and 4) assess transgelin expression in human chronic pancreatitis sections. Culture of PSCs on different matrices significantly affected their gene expression pattern. 146, 619, and 432 genes, respectively, were differentially expressed (P < 0.001) in PSCs cultured on collagen I vs. Matrigel, Matrigel vs. plastic, and collagen I vs. plastic. The highest fold change (12.5-fold upregulation) in gene expression in cells on collagen I vs. Matrigel was observed for transgelin (an actin stress fiber-associated protein). Transgelin was significantly increased in activated PSCs vs. quiescent PSCs. Silencing transgelin expression decreased PSC proliferation and also reduced platelet-derived growth factor-induced PSC migration. Notably, transgelin was highly expressed in chronic pancreatitis in stromal areas and periacinar spaces but was absent in acinar cells. These findings suggest that transgelin is a potentially useful target protein to modulate PSC function so as to ameliorate pancreatic fibrosis.

Dynamic Mast Cell–Stromal Cell Interactions Promote Growth of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) exists in a complex desmoplastic microenvironment, which includes cancer-associated fibroblasts [also known as pancreatic stellate cells (PSC)] and immune cells that provide a fibrotic niche that impedes successful cancer therapy. We have found that mast cells are essential for PDAC tumorigenesis. Whether mast cells contribute to the growth of PDAC and/or PSCs is unknown. Here, we tested the hypothesis that mast cells contribute to the growth of PSCs and tumor cells, thus contributing to PDAC development. Tumor cells promoted mast cell migration. Both tumor cells and PSCs stimulated mast cell activation. Conversely, mast cell-derived interleukin (IL)-13 and tryptase stimulated PSC proliferation. Treating tumor-bearing mice with agents that block mast cell migration and function depressed PDAC growth. Our findings suggest that mast cells exacerbate the cellular and extracellular dynamics of the tumor microenvironment found in PDAC. Therefore, targeting mast cells may inhibit stromal formation and improve therapy.

Pancreatitis activates pancreatic apelin-APJ axis in mice

Pancreatitis is classified into acute pancreatitis (AP) and chronic pancreatitis (CP). Apelin, a small regulatory peptide, is the endogenous ligand for the APJ receptor. Apelin and APJ are expressed in the pancreas. The aims of this study were to examine whether apelin influences the inflammatory and fibrosis responses to pancreatitis in mice and to identify mechanisms behind apelin's activities. Supramaximal cerulein induction of AP or CP caused significant (P < 0.05) elevations in pancreatic apelin and APJ expression. Levels declined during the recovery phases. In apelin gene-knockout mice with pancreatitis, pancreatic neutrophil invasion and myeloperoxidase activity were enhanced significantly, and apelin treatment suppressed both. Apelin exposure reduced CP-induced elevations of extracellular matrix-associated proteins. Apelin inhibited PDGF-simulated connective tissue growth factor production and proliferation of pancreatic stellate cells (PSCs). Serum granulocyte colony-stimulating factor and keratinocyte cytokine levels were higher in apelin gene-knockout than wild-type mice with pancreatitis. Apelin reduced AP- and CP-induced elevations in pancreatic NF-κB activation. Together, these findings imply that the pancreatic apelin-APJ system functions to curb the inflammatory and fibrosis responses during pancreatitis. Furthermore, findings suggest that apelin reduces inflammation and fibrosis by reducing neutrophil recruitment and PSC activity. Inhibition of neutrophil invasion may be mediated by reduced keratinocyte cytokine and granulocyte colony-stimulating factor secretion. Apelin-induced reductions in PSC proliferation and connective tissue growth factor production are putative mechanisms underlying apelin's inhibition of extracellular matrix production. The apelin-associated changes in NF-κB binding may be linked to apelin's regulation of pancreatic inflammatory and fibrosis responses during pancreatitis.

ATP release in pancreatic acini and effects on the P2X7 receptor in pancreatic stellate cells

s / Pancreatolog S92 Department of Clinical Chemistry, University of Ulm, Ulm, Germany Department of General Surgery, Ostalb-klinikum Aalen, Aalen, Germany Department of Internal Medicine, University of Ulm, Ulm, Germany Introduction: Factors mediating the effects of pancreatic stellate cells (PSCs) on pancreatic cancer cells (PCCs) have not been clearly identified. Aims: To investigate the role of PSCs in PCCs migration and to identify underlying mechanisms. Materials & methods: Effects of conditioned PSCs supernatant (PSCSN) and exogenous adhesive molecules on the biology of Panc1 and UlaPaCa cells were investigated by modified Boyden chamber assay, adhesion assay and single cell tracking assay, respectively. Integrin expression and focal adhesion kinase (FAK) phosphorylation were assessed by Western blot. Anti-integrin a2/b1 antibodies and FAK inhibitor (PF-573228) were used to demonstrate the involvement of collagen I. Results: PSC-SN dose-dependently induced PCCs trans-migration, mainly by improving adhesion and motility. PSC-SN mediated adhesion was a prerequisite for the stimulation on PCCs migration. As pure chemokines, PSC-SN was not sufficient to stimulate the trans-migration or motility of PCCs. In contrast to poly-L-lysine or fibronectin, collagen I alone showed resembling effects to PSC-SN on PCCs, including polarized morphology, facilitated adhesion, accelerated motility and transmigration. Both PSC-SN and collagen I induced haptokinesis of Panc1 and haptotaxis of UlaPaCa. Anti-integrin a2/b1 antibodies attenuated PSC-SN/collagen I-induced PCCs trans-migration and adhesion. PSC-SN or collagen I constantly enhanced FAK phosphorylation (Tyr397) in PCCs. PF-573228 diminished PSC-SN/collagen I-induced PCCs haptotaxis/ haptokinesis. Conclusion: Collagen I is the major mediator for PSC-SN induced hapto-migration of PCCs. Through collagen I binding to integrin a2b1 on PCCs, FAK signaling pathway is initiated. S-9 Abstract id: 186. ATP release in pancreatic acini and effects on the P2X7 receptor in pancreatic stellate cells Kristian Agmund Haanes, Ivana Novak. Department of Biology, Copenhagen University, Denmark Introduction: ATP is an extracellular signal released from all cells, including pancreatic acini. Released ATP may stimulate the surrounding pancreatic stellate cells (PSC). Aims: Our aim was to characterize whether pancreatitis-associated stimuli induce ATP release from acini and investigate the importance of ATP in regulating proliferation of PSCs. Materials & methods: Mouse acini and AR42J cells were studied and ATP release was detected with luciferase kit. PSCs were isolated from WT and the Pfizer P2X7 KO mouse using a selective attachment method. Proliferation was monitored with BrdU incorporation. Results: The potential pathophysiological stimuli such as mechanical stress, hypotonicity and the bile acid chenodeoxycholate (CDC) all induced ATP release from acini. Alcohol, however, did not. CDC (1 mM) induced significant ATP release up to 20.4 7.4 nM/10^6 cells/ml. Basal ATP release supported PSC proliferation, as it was inhibited by apyrase, an ATP/ADP hydrolytic enzyme. The proliferation rate was lower in P2X7 KO PSCs compared to WT cells. Also the PSC number from P2X7 KO pancreas was 50% lower than from WT ones. Exogenous ATP further stimulated proliferation in a concentration-dependent and100 mM gave max stimulation. ATP in mM concentration was lethal to PSCs, and this effect was absent in P2X7 KO PSC. Basal/stimulated proliferation and cell death could all be inhibited with the P2X7 antagonists. Conclusion: Together, the study shows that ATP release and purinergic signalling should be considered as important factors in pathophysiological processes in pancreas. Importantly, ATP and P2X7 receptors are regulators of PSC proliferation and death, and could be potential targets in pathologies involving pancreatic fibrosis. y 13 (2013) S2–S98

538 Nuclear Translocation of FGFR1 and FGF2 in Pancreatic Stellate Cells Is Necessary for Pancreatic Cancer Cell Invasion

Background: It is now appreciated that, in pancreatic cancer, transformed cells interact with stromal cells, extracellular matrix proteins, and neighboring normal epithelial cells to exploit feedback mechanisms and facilitate tumour progression. Studies have shown that overexpression of FGF2 in pancreatic cancer correlates with poor patient survival and nuclear FGF2 has been observed in pancreatic cancer patient tissue but not in normal patient samples (Yamanka et al 1993). A number of mechanisms have been proposed for the nuclear accumulation of FGFR1 and its ligand; however, the role of intracrine FGF2 signalling in tumour invasion remains poorly understood. We investigated whether nuclear translocation of FGFR1 with FGF2 plays a role in pancreatc stellate cell (PSC) behaviour. Design: FGF2 and FGFR1 RNAi, together with an FGFR inhibitor (PD173074) were used on pancreatic cancer cell lines PSCs and in organotypic cultures to assess their effect on cell behaviour and to assess the effects of blocking FGF signalling on cancer cell behaviour and invasion. Nuclear FGFR1 and FGF2 expression were also studied in vivo. Results: FGF2 and FGFR1 localised to the nucleus in stromal fibroblasts at the invasive front of human pancreatic cancer tissue. In vitro, FGFR1 and FGF2 co-localised to the nucleus in PSCs but not in cancer or normal ductal epithelial cell lines. PSCs but not cancer cells secrete both HMW and LMW FGF2 isoforms. Abolishing nuclear FGFR1 and FGF2 in PSCs using either RNAi or FGFR inhibitor resulted in a significant reduction in cell proliferation which correlated with a G1 cell-cycle block and reduction in the G1 cyclin, cyclin D1. In an organotypic model, nuclear FGFR1 and FGF2 were significantly greater in PSCs invading into the matrix. When treated with FGFR inhibitor, PSCs were unable to invade into the extra-cellular matrix and FGFR1 and FGF2 remained cytoplasmic. Effective blockade of nuclear FGFR1 signalling in PSCs abolished cancer cell invasion. Conclusion: These studies show for the first time that nuclear FGFR1 and FGF2 plays a role in driving pancreatic stellate cell proliferation. Preventing nuclear FGF/FGFR mediated proliferation in PSCs leads to disruption of the tumour microenvironment, thus preventing pancreatic cancer cell invasion.

Su1790 Apigenin Analogs Effectively Suppress Pancreatic Stellate Cell Proliferation and Induce Apoptosis

Su1790 Apigenin Analogs Effectively Suppress Pancreatic Stellate Cell Proliferation and Induce Apoptosis

Abstract LB-101: Targeting pancreatic cancer stroma with Palmatine, a novel compound from herbal supplement.

Abstract Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer related deaths with 5-year survival rates less than 6%. Survival periods with gemcitabine in combination with various agents average over 6 months. Desmoplasia is a striking feature of pancreatic cancer. It is characterized by formation of dense stroma surrounding the tumor. This dense stroma serves as a therapeutic barrier for many chemotherapeutic drugs, in turn decreasing their efficacy. Pancreatic stellate cells (PSCs) have been reported to be involved in the observed desmoplastic reaction. Therefore targeting this stroma can be an alternate approach to pancreatic cancer. Previous studies from our laboratory showed that Nexrutine, a herbal supplement inhibits proliferation of multiple pancreatic cancer cells through modulation of Stat3/NFκB/Cox-2 signaling and reduced the number of animals developing fibrosis in vivo. To the best of our knowledge, the effect of Nexrutine or its active components on pancreatic stroma has not been studied. In this study we evaluated the effect of Nexrutine and Palmatine, an active component of Nexrutine, for its ability to target the stroma. Our studies show for the first time that Palmatine (i) inhibits proliferation of PSCs as well as the tumor cells; (ii) inhibits hedgehog signaling as evidenced by GLI expression and reporter activity; (iii) modulates expression and activity of downstream targets of GLI including Patched1, IKKB-ε and anti-apoptotic protein Survivin. Remarkably Palmatine treatment inhibits invasive ability of PSCs that is associated with reduced levels of COL1A1. Overall this is the first study reporting the ability of Palmatine to modulate Hh signaling and may have utility either alone or in conjunction with Gemcitabine for treatment of pancreatic ductal adenocarcinoma. Supported by NCCAM (AT 005513-01A1 and AT 007448l; APK). Citation Format: Divya Chakravarthy, Jingjing Gong, Rosa F. Hwang, Addanki Pratap Kumar. Targeting pancreatic cancer stroma with Palmatine, a novel compound from herbal supplement. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-101. doi:10.1158/1538-7445.AM2013-LB-101

Prostaglandin E2 regulates pancreatic stellate cell activity via the EP4 receptor.

Pancreatic stellate cells are source of dense fibrotic stroma, a constant pathological feature of chronic pancreatitis and pancreatic adenocarcinoma. We observed correlation between levels of cyclooxygenase 2 (COX-2) and its product prostaglandin E2 (PGE2) and the extent of pancreatic fibrosis. The aims of this study were to delineate the effects of PGE2 on immortalized human pancreatic stellate cells (HPSCs) and to identify the receptor involved. Immunohistochemistry, reverse transcription-polymerase chain reaction and quantitative reverse transcription-polymerase chain reaction were used to assess COX-2, extracellular matrix, and matrix metalloproteinase gene expression. Eicosanoid profile was determined by liquid chromatography-tandem mass spectrometry. Human pancreatic stellate cell proliferation was assessed by MTS assay, migration by Boyden chamber assay, and invasion using an invasion chamber. Transient silencing was obtained by small interfering RNA. Human pancreatic stellate cells express COX-2 and synthesize PGE2. Prostaglandin E2 stimulated HPSC proliferation, migration, and invasion and stimulated expression of both extracellular matrix and matrix metalloproteinase genes. Human pancreatic stellate cells expressed all 4 EP receptors. Only blocking the EP4 receptor resulted in abrogation of PGE2-mediated HPSC activation. Specificity of EP4 for the effects of PGE2 on stellate cells was confirmed using specific antagonists. Our data indicate that PGE2 regulates pancreatic stellate cell profibrotic activities via EP4 receptor, thus suggesting EP4 receptor as useful therapeutic target for pancreatic cancer to reduce desmoplasia.

Apigenin inhibits pancreatic stellate cell proliferation, stimulates apoptosis and inhibits fibrosis associated with chronic pancreatitis

s / Pancreatology 13 (2013) e1–e94 e84 and 37,418, respectively). Higher islet yield correlated with a greater diabetes free rate and a lesser insulin requirement at the following intervals: pre-operative, post-operative and 6 months after IAT. Conclusion: Prior surgery is strongly correlated with the extent of pancreatic fibrosis, islet yield and insulin requirements in CP patients undergoing TP-IAT. The history of prior pancreatic resection and drainage procedures may be used to predict post-operative islet function and help to determine the optimal timing for TP-IAT in CP patients.

Mechanistic Insights into Self-Reinforcing Processes Driving Abnormal Histogenesis During the Development of Pancreatic Cancer

Pancreatic ductal adenocarcinoma, one of the most feared lethal and painful diseases, is increasing in incidence. The poor prognosis of pancreatic ductal adenocarcinoma-affected patients primarily is owing to our inability to develop effective therapies. Mechanistic studies of genetic, epigenetic, and cell-to-cell signaling events are providing clues to molecular pathways that can be targeted in an attempt to cure this disease. The current review article seeks to draw inferences from available mechanistic knowledge to build a theoretical framework that can facilitate these approaches. This conceptual model considers pancreatic cancer as a tissue disease rather than an isolated epithelial cell problem, which develops and progresses in large part as a result of three positive feedback loops: i) genetic and epigenetic changes in epithelial cells modulate their interaction with mesenchymal cells to generate a dynamically changing process of abnormal histogenesis, which drives more changes; ii) the faulty tissue architecture of neoplastic lesions results in unsynchronized secretion of signaling molecules by cells, which generates an environment that is poor in oxygen and nutrients; and iii) the increased metabolic needs of rapidly dividing cells serve as an evolutionary pressure for them to adapt to this adverse microenvironment, leading to the emergence of resistant clones. We discuss how these concepts can guide mechanistic studies, as well as aid in the design of novel experimental therapeutics.

The P2X7 Receptor Supports Both Life and Death in Fibrogenic Pancreatic Stellate Cells

The pancreatic stellate cells (PSCs) have complex roles in pancreas, including tissue repair and fibrosis. PSCs surround ATP releasing exocrine cells, but little is known about purinergic receptors and their function in PSCs. Our aim was to resolve whether PSCs express the multifunctional P2X7 receptor and elucidate how it regulates PSC viability. The number of PSCs isolated from wild type (WT) mice was 50% higher than those from the Pfizer P2X7 receptor knock out (KO) mice. The P2X7 receptor protein and mRNA of all known isoforms were expressed in WT PSCs, while KO PSCs only expressed truncated versions of the receptor. In culture, the proliferation rate of the KO PSCs was significantly lower. Inclusion of apyrase reduced the proliferation rate in both WT and KO PSCs, indicating importance of endogenous ATP. Exogenous ATP had a two-sided effect. Proliferation of both WT and KO cells was stimulated with ATP in a concentration-dependent manner with a maximum effect at 100 µM. At high ATP concentration (5 mM), WT PSCs, but not the KO PSCs died. The intracellular Ca2+ signals and proliferation rate induced by micromolar ATP concentrations were inhibited by the allosteric P2X7 receptor inhibitor az10606120. The P2X7 receptor-pore inhibitor A438079 partially prevented cell death induced by millimolar ATP concentrations. This study shows that ATP and P2X7 receptors are important regulators of PSC proliferation and death, and therefore might be potential targets for treatments of pancreatic fibrosis and cancer.

Genetic Analyses of Heme Oxygenase 1 (HMOX1) in Different Forms of Pancreatitis

BackgroundHeme oxygenase 1 (HMOX1) is the rate limiting enzyme in heme degradation and a key regulator of inflammatory processes. In animal models the course of pancreatitis was ameliorated by up-regulation of HMOX1 expression. Additionally, carbon monoxide released during heme breakdown inhibited proliferation of pancreatic stellate cells and might thereby prevent the development of chronic pancreatitis (CP). Transcription of HMOX1 in humans is influenced by a GT-repeat located in the promoter. As such, HMOX1 variants might be of importance in the pathogenesis of pancreatitis.MethodsThe GT-repeat and SNP rs2071746 were investigated with fluorescence labelled primers and by melting curve analysis in 285 patients with acute pancreatitis, 208 patients with alcoholic CP, 207 patients with idiopathic/hereditary CP, 147 patients with alcoholic liver cirrhosis, and in 289 controls, respectively. GT-repeat analysis was extended to a total of 446 alcoholic CP patients. In addition, we performed DNA sequencing in 145 patients with alcoholic CP, 138 patients with idiopathic/hereditary CP, 147 patients with alcoholic liver cirrhosis, and 151 controls. Exon 3 screening was extended to additional patients and controls.ResultsS- and L-alleles of the GT-repeat, genotypes and alleles of SNP rs2071746 and non-synonymous variants detected by sequencing were found with similar frequencies in all groups.ConclusionsAlthough functional data implicate a potential influence of HMOX1 variants on the pathogenesis of pancreatitis, we did not find any association. As rare non-synonymous HMOX1 variants were found in patients and controls, it is rather unlikely that they will have functional consequences essential for pancreatitis development.

L-cysteine administration attenuates pancreatic fibrosis induced by TNBS in rats by inhibiting the activation of pancreatic stellate cell.

Background and AimsRecent studies have shown that activated pancreatic stellate cells (PSCs) play a major role in pancreatic fibrogenesis. We aimed to study the effect of L-cysteine administration on fibrosis in chronic pancreatitis (CP) induced by trinitrobenzene sulfonic acid (TNBS) in rats and on the function of cultured PSCs.MethodsCP was induced by TNBS infusion into rat pancreatic ducts. L-cysteine was administrated for the duration of the experiment. Histological analysis and the contents of hydroxyproline were used to evaluate pancreatic damage and fibrosis. Immunohistochemical analysis of α-SMA in the pancreas was performed to detect the activation of PSCs in vivo. The collagen deposition related proteins and cytokines were determined by western blot analysis. DNA synthesis of cultured PSCs was evaluated by BrdU incorporation. We also evaluated the effect of L-cysteine on the cell cycle and cell activation by flow cytometry and immunocytochemistry. The expression of PDGFRβ, TGFβRII, collagen 1α1 and α-SMA of PSCs treated with different concentrations of L-cysteine was determined by western blot. Parameters of oxidant stress were evaluated in vitro and in vivo. Nrf2, NQO1, HO-1, IL-1β expression were evaluated in pancreas tissues by qRT-PCR.ResultsThe inhibition of pancreatic fibrosis by L-cysteine was confirmed by histological observation and hydroxyproline assay. α-SMA, TIMP1, IL-1β and TGF-β1 production decreased compared with the untreated group along with an increase in MMP2 production. L-cysteine suppressed the proliferation and extracellular matrix production of PSCs through down-regulating of PDGFRβ and TGFβRII. Concentrations of MDA+4-HNE were decreased by L-cysteine administration along with an increase in GSH levels both in tissues and cells. In addition, L-cysteine increased the mRNA expression of Nrf2, NQO1 and HO-1 and reduced the expression of IL-1β in L-cysteine treated group when compared with control group.ConclusionL-cysteine treatment attenuated pancreatic fibrosis in chronic pancreatitis in rats.

Effects of Hydrogen Sulfide on Rat Pancreatic Stellate Cells

Pancreatic stellate cells (PSCs) play a crucial role during pancreatic fibrosis development. Hydrogen sulfide (H2S) is a recently discovered gaseous transmitter, whose role in PSCs has not been explored yet. In the present study, we examined the effects of sodium hydrosulfide (NaHS), an H2S donor, on rat PSCs and elucidated the mechanisms involved. Primary PSCs were isolated from rat pancreatic tissue. Lactate dehydrogenase and caspase assays were performed to detect cell death. Pancreatic stellate cell proliferation was determined by cell count analyses, bromodeoxyuridine incorporation, and flow cytometry. The role of heme oxygenase-1 (HO-1) was assessed by pharmacological HO inhibition and transfection of HO-1 small interfering RNA. Pancreatic stellate cell migration was determined by a wound healing assay, and PSC contraction was assessed by a gel contraction assay. α-Smooth muscle actin, collagen type I, and fibronectin messenger RNAs were analyzed by real-time polymerase chain reaction. NaHS inhibited PSC proliferation at nontoxic concentrations. This was associated with HO-1-mediated repression of extracellular signal-regulated kinase 1/2 signaling. NaHS suppressed PSC migration and activation as well as extracellular matrix synthesis. The results of the present study indicate that NaHS inhibits key cell functions of PSCs. Administration of H(2)S-releasing compounds might represent a novel strategy in the treatment of pancreatic fibrosis.

Effects of bone marrow mesenchymal stem cells on pancreatic stellate cells of rats with chronic pancreatitis

Objective To observe the effects of pancreatic stellate cells (PSC) of rats with chronic pancreatitis (CP) after injection of the bone marrow mesenchymal stem cells (BMSCs).Methods The 80 SD rats were randomly divided into four groups:control group (20 rats were injected with sterile saline),model group [20 rats were injected with dibutyltin dichloride (DBT) through the tail vein to make CP model ],treatment group (20 rats were injected with allogeneic GFP-MSCs cultured in vitro when the model of CP was established),and sham treatment group (20 rats were injected with equivalent sterile saline when the model of CP was established).The expression of desmin,glial fibrillary acidic protein (GFAP),α-smooth muscle actin (α-SMA),collagen type Ⅰ and Ⅲ,TGF-β1,IL-10 and TNF-α was detected in the rat pancreas of each group.Results In treatment group,the activity of collegen type Ⅰ (0.135±0.030) ng/g,Ⅲ (0.029±0.008) ng/g,TGF-β1 (0.020±0.006) μg/L in PSC was lower than in sham treatment group ( P ＜ 0.05 ),while that of IL-10 (603.799 ± 89.374) g/ml and TNF-α(507.45 ±90.13) μg/L was higher.All of them had no statistically significant difference between sham treatment group and model group ( P ＞ 0.05 ).No obvious exception was observed in control group.Conclusion By controlling cytokines,BMSCs could reduce the activation,proliferation and differentiation of PSC and lower the deposition of extracellular matrix. Key words: Bone marrow mesenchymal stem cells; Chronic pancreatitis; Stellate cells

Bacterial DNA Promotes Proliferation of Rat Pancreatic Stellate Cells Thorough Toll-Like Receptor 9

We hoped to clarify the possible role of CpG DNA as a trigger factor for overt pancreatic inflammation of pancreatic stellate cells (PSCs). Pancreatic stellate cells were isolated from the male Lewis rat. The expression of Toll-like receptor 9 (TLR9) messenger RNA and protein were evaluated by reverse transcription-polymerase chain reaction and immunofluorescent cytochemistry. Internalization of CpG DNA was analyzed by confocal laser scanning microscopy. Pancreatic stellate cells were incubated with CpG DNA, and then cell proliferation and migration were assessed. Constitutive expression of TLR9 occurs at the messenger RNA and protein levels. After several minutes of CpG DNA administration, CpG DNA was observed on the cell membrane surface and in the cytoplasm and found to be translocating into the perinucleus of PSCs. Pancreatic stellate cells migrated and proliferated in dose- and time-dependent manners in response to simulation by CpG DNA. Proliferation of PSCs was observed 3 hours after administration (earlier than platelet-derived growth factor-induced proliferation), suggesting that PSCs respond readily to provide innate immunity. Endosomal acidification inhibitors attenuated CpG DNA-induced signaling, leading to suppression of DNA synthesis by PSCs. Our findings demonstrate that bacterial DNA promotes migration and proliferation of PSCs and suggest that bacterial DNA can initiate and sustain pancreatic inflammation and fibrosis by means of TLR9.

Effects of Octreotide on Activated Pancreatic Stellate Cell-Induced Pancreas Graft Fibrosis in Rats

Of solid organ transplantations, pancreas transplantation is associated with the highest incidence of pancreatic fibrosis in the early post-transplantation period. Activated pancreatic stellate cells (PSCs) are the main source of pancreatic fibrosis. Octreotide is widely used as a prophylactic for postoperative complications in pancreas transplant recipients. Recent studies have shown that it can inhibit liver fibrosis. This study investigated the effect of octreotide in pancreas graft fibrosis in rats. Isolated PSCs from Sprague Dawley rats were co-cultured with different doses of octreotide (1.25, 2.5, 5, 10, 20, and 40 ng/mL). PSC proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 48, 72, and 96 h. The α-smooth muscle actin (α-SMA) and collagen I expressions of PSCs were detected by immunohistochemistry and reverse-transcriptase polymerase chain reaction. Rat heterotopic pancreaticoduodenal transplantation was performed with and without octreotide treatment (0.01 mg/kg). Pancreas grafts were harvested at postoperative d 1, 3, 5, and 7. Hematoxylin-eosin staining, Masson's trichrome staining, and immunohistochemical staining for α-SMA, collagen I, and tumor growth factor-β1 (TGF-β1) were performed. Octreotide at a concentration of >20 ng/mL significantly inhibited PSC activation and proliferation in vitro. Inflammatory infiltration was reduced in the octreotide group in vivo, and the expression levels of α-SMA, collagen I, and TGF-β1 were also lower, with statistic significant difference or not. Masson's trichrome staining showed a decrease in collagen deposition with octreotide treatment. Octreotide effectively inhibits PSC activation and proliferation in vitro, but has a limited inhibitory effect on the development of pancreas graft fibrosis.

CCK1 and CCK2 Receptors Are Expressed on Pancreatic Stellate Cells and Induce Collagen Production

The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-β-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-β, both CCK and gastrin inhibit proliferation in PSC.