Fibrosis is a characteristic feature of chronic pancreatitis. Roles of Pancreatic Stellate Cells in Pancreatic Inflammation and Fibrosis

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2009;7:S48 S54 Roles of Pancreatic Stellate Cells in Pancreatic Inflammation and Fibrosis ATSUSHI MASAMUNE, TAKASHI WATANABE, KAZUHIRO KIKUTA, and TOORU SHIMOSEGAWA Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan Over a decade, there is accumulating evidence that activated pancreatic stellate cells (PSCs) play a pivotal role in the development of pancreatic fibrosis. In response to pancreatic injury or inflammation, quiescent PSCs are transformed (activated) to myofibroblast-like cells, which express -smooth muscle actin. Activated PSCs proliferate, migrate, produce extracellular matrix components, such as type I collagen, and express cytokines and chemokines. Recent studies have suggested novel roles of PSCs in local immune functions and angiogenesis in the pancreas. If the pancreatic inflammation and injury are sustained or repeated, PSC activation is perpetuated, leading to the development of pancreatic fibrosis. In this context, pancreatic fibrosis can be defined as pathologic changes of extracellular matrix composition in both quantity and quality, resulting from perpetuated activation of PSCs. Because PSCs are very similar to hepatic stellate cells, PSC research should develop in directions more relevant to the pathophysiology of the pancreas, for example, issues related to trypsin, nonoxidative alcohol metabolites, and pancreatic cancer. Indeed, in addition to their roles in chronic pancreatitis, it has been increasingly recognized that PSCs contribute to the progression of pancreatic cancer. Very recently, contribution of bone marrow derived cells to PSCs was reported. Further elucidation of the roles of PSCs in pancreatic fibrosis should promote development of rational approaches for the treatment of chronic pancreatitis and pancreatic cancer. Fibrosis is a characteristic feature of chronic pancreatitis (CP) and of the desmoplastic reaction associated with pancreatic cancer. Until recently, however, the molecular mechanisms of pancreatic fibrosis remained largely unknown at least in part as a result of the lack of appropriate in vitro models. In 1998, star-shaped cells in the pancreas, called pancreatic stellate cells (PSCs), were identified and characterized. 1,2 Over a decade, there is accumulating evidence that activated PSCs play a pivotal role in the development of pancreatic fibrosis in CP and in pancreatic cancer. 1 8 Activation of Pancreatic Stellate Cells In normal pancreas, stellate cells are quiescent and can be identified by the presence of vitamin A containing lipid droplets in the cytoplasm. 1 5 PSCs show mainly a periacinar distribution and constitute 4% of all pancreatic cells. 1,2 Expression of the intermediate filament proteins, desmin and glial fibrillary acidic protein (GFAP), is also used as a marker of quiescent PSCs. The expression and activation of GFAP have been confirmed in transgenic GFAP-LacZ mice where 2.2 kilobase of the GFAP promoter activity was associated exclusively with PSCs. 9 The cell functions of quiescent PSCs remain largely unknown, but periacinar localization suggests a role in maintaining pancreatic acinar cells. In addition, their periductal and perivascular localization suggests that quiescent PSCs might play a role in the regulation of ductal and vascular functions in the pancreas. The physiologic consequences of vitamin A storage in PSCs remain unclear, but it might have a role in maintenance of the quiescent state. McCarroll et al 10 showed that retinol and its metabolites inhibited the induction of -smooth muscle actin ( -SMA) expression in quiescent PSCs and induced quiescence in culture-activated PSCs. It is, therefore, tempting to speculate that retinoic acids are involved in the maintenance of a quiescent phenotype through the binding to their nuclear receptors and the regulation of gene expression. In this scenario, the loss of retinoids in the course of PSC activation might not be an epiphenomenon but essential for senescence. In response to pancreatic injury or inflammation, quiescent PSCs undergo morphologic and functional changes to become myofibroblast-like cells, which express -SMA (Figure 1). This step is called activation. The critical regulatory events that induce PSC activation in vivo are likely to be similar, at least in part, to the events that regulate the activation of primary PSCs in culture in vitro. 1 8 Studies of rat and human primary PSCs in culture have identified a variety of soluble factors, such as cytokines (interleukin [IL]-1, IL-6, and tumor necrosis factor [TNF] ) and growth factors (platelet-derived growth factor [PDGF], transforming growth factor [TGF] 1, and activin A), ethanol and its metabolites, oxidative stress, and pressure, as well as extensive changes in the composition and organization of extracellular matrix (ECM), as regulators of PSC activation Potential sources of these activating factors include activated macrophages, platelets, pancreatic acinar cells, ductal cells, and endothelial cells in inflamed pancreas. It has been shown that pancreatic cancer cells can also be a source of PSC-activating factors. 6 8 Importantly, PSCs by themselves are capable of synthesizing cytokines such as TGF- 1, activin A, and IL-1, suggesting the existence of autocrine loops that might Abbreviations used in this paper: BM, bone marrow; CP, chronic pancreatitis; ECM, extracellular matrix; GFAP, glial fibrillary acidic protein; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; PDGF, platelet-derived growth factor; MMP, matrix metalloproteinase; NADPH, nicotinamide adenine dinucleotide phosphate; PSCs, pancreatic stellate cells; SMA, smooth muscle actin; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TLR, Toll-like receptor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor by the AGA Institute /09/$36.00 doi: /j.cgh

2 November 2009 PSCS AND PANCREATIC FIBROSIS S49 Figure 1. Stimulants of PSC activation. Quiescent PSCs undergo morphologic and functional changes to become activated myofibroblast-like cells. In vitro studies have identified a variety of soluble factors such as cytokines and growth factors, ethanol and its metabolites, oxidative stress, and pressure, as well as extensive changes in the composition and organization of ECM, as regulators of PSC activation. During pancreatic injury, inflammation, and pancreatic cancer in vivo, PSCs are likely to be exposed to these stimuli. contribute to the perpetuation of PSC activation after stimulation by an initial exogenous signal, thereby promoting the development of fibrosis Functions of Pancreatic Stellate Cells On activation, PSCs lose lipid droplets, more actively proliferate, migrate, produce ECM components, and secrete proinflammatory cytokines and chemokines. 1 8,19 Cytokines and growth factors produced by acinar cells, inflammatory cells, platelets, ductal cells, endothelial cells, cancer cells, and PSCs by themselves could activate PSCs and induce these cell responses in paracrine and autocrine manners. 1 8 Accumulating evidence supports major roles for PDGF, which induces proliferation and migration of PSCs, and TGF- 1, which induces PSCs to express -SMA and ECM proteins, as mediators of the persistently activated and profibrogenic phenotypes of PSCs In addition, recent studies have revealed that PSCs have a variety of cell functions (Table 1). Not only do PSCs produce ECM components, they also produce matrix-degrading enzymes of the matrix metalloproteinases (MMPs) family and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). PSCs have been shown to secrete MMP-2, MMP-9, and MMP-13 and to express TIMP-1 and TIMP Thus, PSCs might be involved in the maintenance of normal tissue architecture by regulating ECM turnover. In this scenario, resolution of cerulein-induced pancreatitis in mice involves transient activation of PSCs and deposition of ECM proteins, as well as transient up-regulation of MMPs and TIMPs. 21 On the other hand, MMP-2 produced by PSCs might contribute to the progression of pancreatic cancer. 22 The increased expression of the cytoskeletal protein -SMA confers increased contractile potential, which is further enhanced by endothelin As mentioned, PSCs are located around the ductal and vascular structures 1 5 ; PSC contraction could regulate vascular and ductal tone in the pancreas. PSCs have the ability to produce a wide variety of cytokines and growth factors. PSCs produce IL-1, IL-6, TNF-, TGF- 1, and PDGF-BB, all of which contribute to perpetuation of PSC activation. 3 5,11 19,24,25 Chemokines (IL-8, monocyte chemoattractant protein [MCP]-1, and RANTES) produced by PSCs contribute to the recruitment of inflammatory cells to the inflamed pancreas. 24,25 Expression of cell adhesion molecules, such as intercellular adhesion molecule-1 in PSCs, also contributes to the adhesion of recruited inflammatory cells. 26 Very recently, we and others have shown that PSCs express Toll-like receptors (TLRs), proteins involved in the activation of innate immunity. 27,28 PSCs express TLR2, which recognizes pathogenassociated molecular patterns of gram-positive bacteria, and TLR4, which recognizes lipopolysaccharides of gram-negative bacteria. PSCs also express TLR3, which recognizes doublestranded RNA produced during viral replication, and TLR5, which recognizes flagellin, the major component of bacterial flagella. In addition, PSCs endocytose and phagocytose foreign bodies, necrotic debris, and aged polymorphonuclear cells, suggesting that PSCs might have a role in the local immune functions in the pancreas. 28,29 Thus, it is likely that PSCs play a macrophage-like role in the pancreas, comparable to the role of Kupffer cells in the liver. PSCs might contribute to organ restitution and homeostasis by engulfing pancreatic acinar cells undergoing apoptosis and necrosis. 30 A novel function we recently identified in PSCs is related to angiogenesis. 31 PSCs constitutively produce vascular endothelial growth factor (VEGF), and its generation is increased by hypoxia. In addition to VEGF, PSCs express several angiogenesis-regulating molecules including VEGF receptors (Flt-1 and Flk-1), angiopoietin-1 and its receptor Tie-2, and vasohibin-1. Conditioned media of hypoxia-treated PSCs induced angiogenesis in vitro and in vivo; it increased tube formation on Matrigel (BD Biosciences, Franklin Lakes, NJ) and directed vessel formation in nude mice (Figure 2). 31 A significant association between fibrosis, angiogenesis, and higher VEGF expression has been reported in pancreatic cancer and in CP. 32 Thus, PSCs might play profibrogenic and proangiogenic roles during the development of pancreatic fibrosis, where they are subjected to hypoxia. Although further in vivo studies are needed for a more detailed characterization of this scenario, these findings suggest a novel mechanism linking hypoxia, inflammatory responses, angiogenesis, and fibrosis in the pancreas. Table 1. Responses of PSC to Stimulation and Activation -SMA expression Proliferation ECM production (type I, type III collagen, etc) Cytokine, chemokine production (IL-8, MCP-1, etc) Adhesion molecule (ICAM-1) expression Migration/chemotaxis Contractility Matrix degradation (MMPs expression) TLRs expression Endocytosis and phagocytosis Angiogenic responses

3 S50 MASAMUNE ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 11S Figure 2. Conditioned media of hypoxia-treated PSCs induced angiogenesis in vitro and in vivo. (A) Human umbilical vein endothelial cells (HUVECs) were added to each well onto solidified ECM gel in 600 L of serum-free medium or of conditioned media of hypoxia-treated PSCs (PSC-CM). After 18-hour incubation, endothelial cell tube formation was assessed under light microscopy. Original magnification, 4 objective. (B) Pre-chilled, semi-closed silicone cylinders (angioreactors) were filled with 20 L of a basement membrane extract premixed with a control serum-free medium or 10-fold concentrated conditioned media from hypoxia-treated PSCs. After forming a gel, angioreactors were implanted subcutaneously in the dorsal flanks of nude mice. After 14 days, angioreactors were removed from mice, and vessel formation was assessed. Arrows indicate the direction of vessel formation. Postactivation Events After the initiation of activation, PSCs might have 2 fates (Figure 3). If the inflammation and injury are sustained or repeated, PSC activation is perpetuated, leading to the development of pancreatic fibrosis. By contrast, if the inflammation and injury are limited, PSCs might undergo apoptosis or revert to quiescence. In this case, fibrosis will not develop. From this point of view, pancreatic fibrosis can be defined as pathologic changes of ECM composition in the pancreas in both quantity and quality, resulting from persistent PSC activation. To develop pancreatic fibrosis, repeated and persistent injury and inflammation are important. This concept is in agreement with the belief that repeated episodes of acute pancreatitis lead to the development of pancreatic fibrosis (necrosis-fibrosis sequence). 33,34 In the case of pancreatic cancer, neoplastic cells initiate and perpetuate PSC activation. 6 8 Interaction Between Pancreatic Stellate Cells and Cancer Cells Because PSCs are morphologically and functionally very similar to hepatic stellate cells, the major effector cells in liver Figure 3. Postactivation events. After initiation of activation, PSCs might have 2 fates. If inflammation and injury are sustained or repeated, PSC activation is perpetuated, leading to development of pancreatic fibrosis. However, if inflammation and injury are limited, PSCs might undergo apoptosis or revert to quiescent stage. In the latter scenario, fibrosis will not develop.

4 November 2009 PSCS AND PANCREATIC FIBROSIS S51 Figure 4. Interactions between PSCs and cancer cells promote progression of pancreatic cancer. In vitro studies have shown that conditioned media of pancreatic cancer cells induced proliferation, production, and degradation of ECM and angiogenesis in PSCs, at least in part through PDGF, TGF, and fibroblast growth factor-2. Conversely, conditioned media of PSCs stimulated cancer cell proliferation and migration and protected them from radiation and gemcitabine-induced apoptosis, in part through the action of PDGF. In addition, PSCs can create a tumor-supportive microenvironment enriched with matricellular proteins that include periostin, galectins, connective tissue growth factor, cysteine-rich acidic secreted protein, and fibrinogen. fibrosis, it is particularly interesting to see whether the 2 populations differ in content or function. DNA array study showed that only 29 of 23,000 genes (0.1%) were different between PSCs and hepatic stellate cells. 35 It would seem that research of PSCs should develop in directions more relevant to the pathophysiology of the pancreas, for example, responses to trypsin, nonoxidative alcohol metabolites, and pancreatic cancer. 36,37 Indeed, the interaction between PSCs and pancreatic cancer cells is receiving increasing attention. 6 8,22,38 41 There is accumulating evidence that PSCs promote the progression of pancreatic cancer. For example, it has been shown that conditioned media of pancreatic cancer cells induce proliferation, ECM production, and TIMP-1 production in PSCs. These responses, at least in part, are mediated by fibroblast growth factor-2, TGF- 1, and PDGF. 6 8,38 On the other hand, conditioned media of PSCs increased proliferation, invasion, and colony formation in pancreatic cancer cells. 6 8 Conditioned media of PSCs also protected pancreatic cancer cells from H 2 O 2 -, radiation- and gemcitabine-induced apoptosis. 7,8 In addition to these cytokine-mediated mechanisms, we and others have shown that PSCs create a tumor-supportive microenvironment by producing matricellular proteins The term matricellular has been applied to a group of extracellular proteins that do not contribute directly to the formation of structural elements in vertebrates but serve to modulate cell-matrix interactions and cellular functions. 45 Although not being structurally related, matricellular proteins regulate similar biologic functions during embryonic development, tissue injury, and cancer development mainly by promoting adhesion, migration, and survival of cells. 45 Significant up-regulation of matricellular proteins including galectin-1, periostin, connective tissue growth factor, tenascin-c, secreted acidic proteins rich in cysteine, and fibrinogen was demonstrated in the stromal tissues of pancreatic cancer and CP, as well as in activated PSCs The abilities of these matricellular proteins to stimulate PSCs and pancreatic cancer cells have been shown. Thus, matricellular proteins induced proliferation, migration, production of cytokines and ECM, and angiogenic responses in PSCs In addition, matricellular proteins induce cancer cell proliferation and migration. Importantly, expression of these matricellular proteins has been shown to correlate with poor survival of pancreatic cancer patients. 39,41 Thus, these matricellular proteins might directly contribute to the progression of pancreatic cancer by stimulating cancer cell activities and indirectly by creating a tumor-supportive microenvironment through sustained fibrogenic stellate cell activity (Figure 4). To support these in vitro findings, in vivo studies have shown that coinjection of cancer cells with PSCs increased tumor size in a subcutaneous xenograft model 6 and tumor incidence, growth, and metastasis in orthotopic models of pancreatic cancer 7,8 (Figure 5). Although controversial, these recent studies support the concept that the desmoplastic responses created by the Figure 5. PSCs support the growth of cancer cells in vivo. PC-1 pancreatic cancer cells were injected subcutaneously into nude mice either alone (left side of the back) or together with PSCs (right side). After 4 weeks, the size of subcutaneous tumor was greater if PSCs were injected together with cancer cells (right side) (unpublished observations: Masamune A, et al).

5 S52 MASAMUNE ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 11S cancer cells to PSC interactions favor the progression of pancreatic cancer. Origin of Pancreatic Stellate Cells Although PSCs and hepatic stellate cells might exhibit functional differences, the 99.9% identity at the mrna level suggests that PSCs and hepatic stellate cells might have a common origin. Activated PSCs have been thought to arise from transformation of quiescent PSCs that reside in the pancreas. 1 5 However, studies with sex-mismatched bone marrow (BM) transplantation from a transgenic male mouse carrying enhanced green fluorescent protein to female wild-type mice suggested that BM-derived cells also contribute to the population of PSCs. Thus, after induction of CP with repeated injections of the cholecystokinin analogue, cerulein, BM-derived cells accounted for about 5% 20% of total -SMA positive activated PSCs 46 (Watanabe T et al, unpublished data). Interestingly, even before the induction of pancreatitis, we could identify desmin-positive, BM-derived cells in the pancreas (Figure 6). Indeed, we found that BM-derived cells accounted for about 8.7% of the desmin (a marker of PSCs)-positive cells in the pancreas at 8 weeks after BM transplantation. Furthermore, we were able to isolate BM-derived, green fluorescent protein-positive cells containing lipid droplets in the cytoplasm, further supporting the conclusion that BM contributed to the population of quiescent PSCs. Thus, BM-derived cells contribute to both quiescent and activated PSC populations in mice. Contributions from fibrocytes, endothelial cells, through the endothelial-to-mesenchymal transition and epithelial cells, and through epithelial-to-mesenchymal transition to the population of PSCs are also likely but remain to be conclusively demonstrated. Antifibrosis Therapies Targeting Pancreatic Stellate Cells Because activated PSCs play a pivotal role in the development of pancreatic fibrosis, factors that control their cellular functions could provide potential targets for the treatment of pancreatic fibrosis and inflammation. 47 Antifibrosis treatment strategies that target PSCs include (1) inhibiting activation of quiescent PSCs, (2) controlling cell functions in PSCs, (3) anticytokine and anti growth factor therapies, (4) induction of apoptosis and reversion to quiescence in PSCs, and (5) removal of perpetuating factors (inflammation and alcohol abuse). Beneficial effects of several agents, which inhibit the activation and PSC functions in vitro, have been shown in experimental models of CP in vivo The agents include peroxisome proliferator-activated receptor- ligands, angiotensin receptor blockers, angiotensin-converting enzyme inhibitors, antioxidants including polyphenols, serine-protease inhibitors, and an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase Thus, diphenyleneiodonium inhibited PDGF-induced proliferation, IL-1 induced chemokine production, and expression of -SMA and collagen in PSCs in vitro. 54 Diphenyleneiodonium also inhibited the development of pancreatic fibrosis in male Wistar Bonn/Kobori rats and in rats with dibutyltin dichloride-induced CP. 54 Similarly, camostat mesilate, an oral protease inhibitor used to treat patients with CP, inhibited proliferation and MCP-1 production in PSCs in vitro. 50 Camostat mesilate also inhibited the development of pancreatic fibrosis in rats with dibutyltin dichloride induced CP. 50 Because reactive oxygen species mediate activation and cell functions in PSCs, antioxidants such as plant polyphenols are candidates for the development of antifibrosis therapy targeting PSCs It remains unknown whether antifibrosis therapies might be useful for the treatment of pancreatic cancer through the destruction of a cancer-supportive microenvironment. Concluding Remarks Pancreatic fibrosis can be defined as pathologic changes of ECM composition in both quantity and quality, resulting from perpetual activation of PSCs. In addition to their roles in CP, it is increasingly recognized that PSCs contribute to the progression of pancreatic cancer. In this context, a major investigative goal will be to determine whether modulators of stellate cell activities can be developed that will prove useful for the treatment of pancreatic cancer as well as of CP. References Figure 6. BM-derived cells contributed to the population of activated PSCs in pancreatic fibrosis in mice. Eight weeks after cross-gender BM transplantation, CP was induced by 6 intra-abdominal injections of cerulein at 1-hour intervals, 3 days each week, for total of 6 weeks in recipient female mice. The pancreata were removed 6 weeks after the beginning of cerulein treatment. Immunofluorescent staining for -SMA (shown in red) was performed, along with fluorescence in situ hybridization for the Y chromosome. Nuclei were counterstained with DAPI (blue). Arrows indicate representative -SMA /Y cells. Original magnification, 400 (unpublished observations: Watanabe T, et al). 1. Apte MV, Haber PS, Applegate TL, et al. Periacinar stellateshaped cells in rat pancreas: identification, isolation and culture. Gut 1998;43: Bachem MG, Schneider E, Gross H, et al. Identification, culture, and characterization of pancreas stellate cells in rats and humans. Gastroenterology 1998;115: Omary MB, Lugea A, Lowe AW, et al. The pancreatic stellate cell: a star on the rise in pancreatic diseases. J Clin Invest 2007;117: Shimizu K. Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis. J Gastroenterol 2008;43: Masamune A, Shimosegawa T. Signal transduction in pancreatic stellate cells. J Gastroenterol 2009;44: Bachem MG, Schünemann M, Ramadani M, et al. Pancreatic

6 November 2009 PSCS AND PANCREATIC FIBROSIS S53 carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology 2005;128: Hwang RF, Moore T, Arumugam T, et al. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 2008;68: Vonlaufen A, Joshi S, Qu C, et al. Pancreatic stellate cells: partners in crime with pancreatic cancer cells. Cancer Res 2008; 68: Ding Z, Maubach G, Masamune A, et al. Glial fibrillary acidic protein promoter targets pancreatic stellate cells. Dig Liver Dis 2009;41: McCarroll JA, Phillips PA, Santucci N, et al. Vitamin A inhibits pancreatic stellate cell activation: implications for treatment of pancreatic fibrosis. Gut 2006;55: Luttenberger T, Schmid-Kotsas A, Menke A, et al. Platelet-derived growth factors stimulate proliferation and extracellular matrix synthesis of pancreatic stellate cells: implications in pathogenesis of pancreas fibrosis. Lab Invest 2000;80: Schneider E, Schmid-Kotsas A, Zhao J, et al. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells. Am J Physiol Cell Physiol 2001;281: C532 C Mews P, Phillips P, Fahmy R, et al. Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut 2002;50: Ohnishi N, Miyata T, Ohnishi H, et al. Activin A is an autocrine activator of rat pancreatic stellate cells: potential therapeutic role of follistatin for pancreatic fibrosis. Gut 2003;52: Apte MV, Haber PS, Darby SJ, et al. Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis. Gut 1999;44: Apte MV, Phillips PA, Fahmy RG, et al. Does alcohol directly stimulate pancreatic fibrogenesis? Gastroenterology 2000;118: Masamune A, Kikuta K, Satoh M, et al. Alcohol activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells. J Pharmacol Exp Ther 2002;302: Watanabe S, Nagashio Y, Asaumi H, et al. Pressure activates rat pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2004;287:G1175 G Masamune A, Kikuta K, Satoh M, et al. Differential roles of signaling pathways for proliferation and migration of rat pancreatic stellate cells. Tohoku J Exp Med 2003;199: Phillips PA, McCarroll JA, Park S, et al. Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover. Gut 2003;52: Lugea A, Nan L, French SW, et al. Pancreas recovery following cerulein-induced pancreatitis is impaired in plasminogen-deficient mice. Gastroenterology 2006;131: Schneiderhan W, Diaz F, Fundel M, et al. Pancreatic stellate cells are an important source of MMP-2 in human pancreatic cancer and accelerate tumor progression in a murine xenograft model and CAM assay. J Cell Sci 2007;120: Masamune A, Satoh M, Kikuta K, et al. Endothelin-1 stimulates contraction and migration of rat pancreatic stellate cells. World J Gastroenterol 2005;11: Masamune A, Kikuta K, Satoh M, et al. Ligands of peroxisome proliferator-activated receptor-gamma block activation of pancreatic stellate cells. J Biol Chem 2002;277: Andoh A, Takaya H, Saotome T, et al. Cytokine regulation of chemokine (IL-8, MCP-1, and RANTES) gene expression in human pancreatic periacinar myofibroblasts. Gastroenterology 2000; 119: Masamune A, Sakai Y, Kikuta K, et al. Activated rat pancreatic stellate cells express intercellular adhesion molecule-1 (ICAM-1) in vitro. Pancreas 2002;25: Vonlaufen A, Xu Z, Daniel B, et al. Bacterial endotoxin: a trigger factor for alcoholic pancreatitis? evidence from a novel, physiologically relevant animal model. Gastroenterology 2007;133: Masamune A, Kikuta K, Watanabe T, et al. Pancreatic stellate cells express Toll-like receptors. J Gastroenterol 2008;43: Shimizu K, Kobayashi M, Tahara J, et al. Cytokines and peroxisome proliferator-activated receptor gamma ligand regulate phagocytosis by pancreatic stellate cells. Gastroenterology 2005; 128: Tahara J, Shimizu K, Shiratori K. Engulfment of necrotic acinar cells by pancreatic stellate cells inhibits pancreatic fibrogenesis. Pancreas 2008;37: Masamune A, Kikuta K, Watanabe T, et al. Hypoxia stimulates pancreatic stellate cells to induce fibrosis and angiogenesis in pancreatic cancer. Am J Physiol Gastrointest Liver Physiol 2008; 295:G709 G Kuehn R, Lelkes PI, Bloechle C, et al. Angiogenesis, angiogenic growth factors, and cell adhesion molecules are upregulated in chronic pancreatic diseases: angiogenesis in chronic pancreatitis and in pancreatic cancer. Pancreas 1999;18: Comfort H, Gambill E, Baggenstoss A. Chronic relapsing pancreatitis: a study of 29 cases without associated disease of the biliary or gastrointestinal tract. Gastroenterology 1946;6: Klöppel G, Maillet B. The morphological basis for the evolution of acute pancreatitis into chronic pancreatitis. Virchows Arch A Pathol Anat Histopathol 1992;420: Buchholz M, Kestler HA, Holzmann K, et al. Transcriptome analysis of human hepatic and pancreatic stellate cells: organ-specific variations of a common transcriptional phenotype. J Mol Med 2005;83: Masamune A, Kikuta K, Satoh M, et al. Protease-activated receptor-2-mediated proliferation and collagen production of rat pancreatic stellate cells. J Pharmacol Exp Ther 2005;312: Masamune A, Satoh A, Watanabe T, et al. Effects of ethanol and its metabolites on human pancreatic stellate cells. Dig Dis Sci 2009 Jan 23 [Epub ahead of print]. 38. Yoshida S, Yokota T, Ujiki M, et al. Pancreatic cancer stimulates pancreatic stellate cell proliferation and TIMP-1 production through the MAP kinase pathway. Biochem Biophys Res Commun 2004;323: Erkan M, Kleeff J, Gorbachevski A, et al. Periostin creates a tumor-supportive microenvironment in the pancreas by sustaining fibrogenic stellate cell activity. Gastroenterology 2007;132: Kanno A, Satoh K, Masamune A, et al. Periostin, secreted from stromal cells, has biphasic effect on cell migration and correlates with the epithelial to mesenchymal transition of human pancreatic cancer cells. Int J Cancer 2008;122: Mantoni TS, Schendel RR, Rödel F, et al. Stromal SPARC expression and patient survival after chemoradiation for non-resectable pancreatic adenocarcinoma. Cancer Biol Ther 2008;7: Gao R, Brigstock DR. Connective tissue growth factor (CCN2) in rat pancreatic stellate cell function: integrin alpha5beta1 as a novel CCN2 receptor. Gastroenterology 2005;129: Masamune A, Satoh M, Hirabayashi J, et al. Galectin-1 induces chemokine production and proliferation in pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2006;290:G729 G Masamune A, Kikuta K, Watanabe T, et al. Fibrinogen induces cytokine and collagen production in pancreatic stellate cells. Gut 2009;58:

7 S54 MASAMUNE ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 11S 45. Bornstein P, Sage EH. Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol 2002;14: Marrache F, Pendyala S, Bhagat G, et al. Role of bone marrowderived cells in experimental chronic pancreatitis. Gut 2008;57: Talukdar R, Tandon RK. Pancreatic stellate cells: new target in the treatment of chronic pancreatitis. J Gastroenterol Hepatol 2008;23: Shimizu K, Shiratori K, Hayashi N, et al. Thiazolidinedione derivatives as novel therapeutic agents to prevent the development of chronic pancreatitis. Pancreas 2002;24: Kuno A, Yamada T, Masuda K, et al. Angiotensin-converting enzyme inhibitor attenuates pancreatic inflammation and fibrosis in male Wistar Bonn/Kobori rats. Gastroenterology 2003;124: Gibo J, Ito T, Kawabe K, et al. Camostat mesilate attenuates pancreatic fibrosis via inhibition of monocytes and pancreatic stellate cells activity. Lab Invest 2005;85: Masamune A, Suzuki N, Kikuta K, et al. Curcumin blocks activation of pancreatic stellate cells. J Cell Biochem 2006;97: Masamune A, Satoh M, Kikuta K, et al. Ellagic acid blocks activation of pancreatic stellate cells. Biochem Pharmacol 2005; 70: Suzuki N, Masamune A, Kikuta K, et al. Ellagic acid inhibits pancreatic fibrosis in male Wistar Bonn/Kobori rats. Dig Dis Sci 2009;54: Masamune A, Watanabe T, Kikuta K, et al. NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2008;294:G99 G108. Reprint requests Address requests for reprints to: Atsushi Masamune, MD, PhD, Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai , Japan. amasamune@m.tains.tohoku.ac.jp; fax: (81) Conflicts of interest The authors disclose no conflicts. Funding Supported in part by Grant-in-Aid from Japan Society for the Promotion of Science (A.M.), by the Pancreas Research Foundation of Japan (A.M.), by the Kanae Foundation for Life and Socio-Medical Science (A.M.), by the Uehara Memorial Foundation (A.M.), and by the Research Committee of Intractable Pancreatic Diseases (Principal investigator, T.S.) provided by the Ministry of Health, Labour, and Welfare of Japan.