Gut Online First, published on November 24, 2011 as 10.1136/gutjnl-2011-301220 Leading article Gut: first published as 10.1136/gutjnl-2011-301220 on 24 November 2011. Downloaded from extending along the base of adjacent StellaTUM: current consensus acinar cells similar to that of in the mammary gland. In health, PSC exist in their quiescent phenotype and exhibit and discussion on pancreatic the presence of abundant vitamin A-containing lipid droplets in their cyto- stellate cell research plasm. It is estimated that quiescent PSC form 4e7% of all parenchymal cells in the 1 2 3 normal .45During pancreatic Mert Erkan, Guido Adler, Minoti V Apte, injury, resident PSC transform into an Max G Bachem,4 Malte Buchholz,5 So¨nke Detlefsen,6 activated phenotype that secretes exces- 7 1 5 sive amounts of the Irene Esposito, Helmut Friess, Thomas M Gress, (ECM) proteins that comprise fibrous 4 8 9 tissue. Recent evidence suggests that Hans-Joerg Habisch, Rosa F Hwang, Robert Jaster, a small proportion of activated PSC may 1 7 10 also be derived from circulating Jo¨rg Kleeff, Gu¨nter Klo¨ppel, Claus Kordes, marrow (BM)-derived cells that home to 8 11 Craig D Logsdon, Atsushi Masamune, the pancreas during pancreatic injury. 1 12 Christoph W Michalski, Junseo Oh, STELLATE CELL SYSTEM AND THE ORIGIN 3 13 OF PSC AND HSC Phoebe A Phillips, Massimo Pinzani, In the human body, the stellate cell system Carolin Reiser-Erkan,1 Hidekazu Tsukamoto,14 consists of retinoid-storing cells in various 3 organs, including the , pancreas, lung, Jeremy Wilson kidney, intestine, spleen, adrenal gland, ductus deferens and vocal cords showing a perivascular location with a distribution 67 The field of pancreatic stellate cell (PSC) into elucidating the biology and function of typical of a . However, the origin biology is very young, as the essential in- these cells. It is now well established that of stellate cells is still being debated. 8e10 11 12 vitro tools to study these cells (ie, methods PSC are responsible for producing the Mesenchymal, endodermal as well 13e15 to isolate and culture PSC) were only stromal reaction (fibrosis) of two major as neuroectodermal origins are developed as recently as in 1998. Nonethe- diseases of the pancreasdchronic pancrea- suggested. Neuroectoderm and mesoderm less, there has been an exponential increase titis and . Despite expo- were considered as two potential origins of d http://gut.bmj.com/ in research output in this field over the past nentially increasing data, the methods for HSC these cells and PSC share numerous decade, with numerous research groups studying PSC remain variable. Although characteristics as indicated by morpholog- around the world focusing their energies within individual laboratories methods are ical, functional and gene expression fi consistent, different methodologies used by studies. Expression pro ling of PSC, HSC fi various research groups make it difficult to and broblasts has demonstrated that PSC 1Department of Surgery, Klinikum rechts der Isar, TU and HSC are distinctly different from 2 compare results and conclusions. This Mu¨nchen, Munich, Germany; Department of Internal fibroblasts, but share many homologies

article is not a review article on the func- on September 27, 2021 by guest. Protected copyright. Medicine, Johannes Gutenberg-Universita¨t Mainz, including the expression of genes related to Mainz, Germany; 3Faculty of Medicine, The University of tions of PSC. Instead, members of the New South Wales, Sydney, New South Wales, Pancreatic Star Alliance (http://www. ECM proteins, contractility, retinoid Australia; 4Department of Clinical Chemistry, pancreaticstaralliance.com) discuss here metabolism (although lower retinoid 5 Universita¨tsklinikum Ulm, Ulm, Germany; Department of and consolidate current knowledge, to content in PSC compared with HSC) and Internal Meidicine, Universita¨tsklinikum Marburg, 16 6 outline and delineate areas of consensus or growth factors. In fact, both cells (in Marburg, Germany; Department of Pathology, Odense fi University Hospital, Odense, Denmark; 7Institute of otherwise (eg, with regard to methodolog- comparison with broblasts) have more 17 Pathology, Klinikum rechts der Isar, TU Mu¨nchen, ical approaches) and, more importantly, to similarities than differences. It is there- 8 Munich, Germany; Department of Surgical Oncology, identify essential directions for future fore possible that HSC and PSC share University of Texas MD Anderson Cancer Center, 9 research. a common origin. Houston, Texas, USA; Department of Internal Medicine, The neuroectoderm proposal for the University of Rostock, Rostock, Germany; 10Department of Gastroenterology, Hepatology and Infectious PANCREATIC STELLATE CELLS origins of HSC was refuted by a study by 10 Diseases, Universita¨tsklinikum Du¨sseldorf, Du¨sseldorf, Hepatic stellate cells (HSC) were first Cassiman et al, who used a genetic cell Germany; 11Division of Gastroenterology, Tohoku described by Karl von Kupffer in 1876; lineage mapping technique with University Graduate School of Medicine, Sendai, Miyagi, flox 12 however, similar cells in the pancreas were Rosa26YFP mice crossed with mice Tohoku, Japan; Laboratory of Cellular Oncology, Korea fi 1e3 University Graduate School of Medicine, Seoul, South rst observed in the 1980s. In 1998, expressing Cre under the control of 4 5 Korea; 13Department of Internal Medicine, University of Apte et al and Bachem et al isolated and the neural crest-specific Wnt1 promoter/ Florence, Florence, Italy; 14Southern California Research cultured PSC.45In the normal pancreas, enhancer. Definitive novel evidence for Center for ALPD and , Keck School of Medicine PSC are located in close proximity to the the mesodermal origin of HSC has of the University of Southern California, Los Angeles, basal aspect of pancreatic acinar cells. In recently been presented by Asahina and California, USA sections immunostained for the marker colleagues818with the use of the meso- Correspondence to Dr Mert Erkan, Department of fi General Surgery, Technische Universita¨tMunchen, (a cytoskeletal protein), quiescent derm-speci c MesP1Cre. A conditional ¨ CreERT Klinikum rechts der Isar, 81675 Munich, Germany; PSC can be seen as cells with a central cell cell lineage analysis using Wt1 / fl fl [email protected] body and long cytoplasmic projections Rosa26LacZ ox or ROSA26mTmG ox mice,

CopyrightErkan M, Adler Article G, Apte author MV, et al. Gut(or(2011). their doi:10.1136/gutjnl-2011-301220 employer) 2011. Produced by BMJ Publishing Group Ltd (& BSG) under licence.1of7 Leading article Gut: first published as 10.1136/gutjnl-2011-301220 on 24 November 2011. Downloaded from revealed Wt1-positive septum transversum as nestin and CD133, which are known to colleagues4 from Professor Wilson’s labo- giving rise to mesothelial cells, submeso- be expressed on somatic stem cells.24 ratory in 1998. This method took advan- thelial cells, HSC and perivascular mesen- However, before HSC can be classified as tage of the known vitamin A content chymal cells during liver development.8 stem cells, essential characteristics of stem (stored in cytoplasmic lipid droplets) of That study also demonstrated Wt1-posi- cells should be defined and verified in stel- PSC, which allowed the cells to be sepa- tive mesothelial/submesothelial cells late cells. Briefly, stem cells are undifferen- rated by a single density gradient method migrating inward from the liver surface to tiated cells with the potential to proliferate using nycodenz. More recently, a similar generate liver mesenchymal cells including and to undergo developmental processes. method (albeit with some modifications) HSC. Similar lineage tracing techniques More specifically, stem cells: (1) express was used to isolate quiescent PSC from need to be used to determine the exact genes required for the inhibition or induc- normal human pancreas.28 The gradients origin of PSC. tion of cell development; (2) maintain their can be developed not only with nycodenz characteristics in a special microenviron- but also with other colloids such as ROLE OF PSC IN AND ment (stem cell niche); (3) are normally percoll, iohexol, iodixanol, optiprep. The REGENERATION quiescent, but can be activated on demand; average yield of cells from a rat pancreas is The origin and fate of stellate cells in the (4) proliferate/migrate when activated; and approximately 3 million cells per gram of context of injury and regeneration are also (5) can differentiate into effector cells pancreas. The yield of PSC is much lower a matter of ongoing debate. Regarding (plasticity) or influence the developmental from human pancreas due to limited the contribution of BM and epithelial fate of other cells. Through these mecha- tissue availability and due to the higher mesenchymal transition (EMT) of acinar nisms stem cells participate in ontogenesis, amount of fatty tissue surrounding the cells to the PSC population, the data are regeneration or reproduction of organisms. resected pancreas (compared with rat limited and restricted to studies in mice. In Furthermore, stem cells are transplantable pancreas), which can impede tissue diges- 2006, it was reported that BM is a source and can survive for a long time within host tion. PSC can also be isolated from of myofibroblast-like cells in fibrotic tissues. First steps were made to unravel cancerous or fibrotic human pancreas, as liver tissue, but the involvement of these these characteristics in HSC of rodents. well as normal rat pancreas, using the cells in the progression of liver fibrosis Genes required for stemness and develop- outgrowth method first described by remains questionable as their contribution mental processes are expressed by HSC, Bachem and colleagues5 from Professor to synthesis appears to be which preserve their quiescent state within Adler’s laboratory in 1998. The PSC limited.19 20 Recent studies have now also their niche, the space of Dissé.24 25 More- obtained by the outgrowth method addressed the role of BM-derived cells in over, HSC are quiescent in normal liver, but comprise cells of different age due to pancreatic regeneration and fibrosis.21 22 become activated especially during stem temporal differences in their growing out Using a similar experimental approach, cell-based liver repair. The gene expression from fibrotic tissue blocks. Both methods namely the transfer of green fluorescent of different cell types such as yield a mixed population of stromal cells, protein (GFP)-expressing BM cells to is inducible in activated HSC by cytokine which are globally named PSC but have lethally irradiated small rodents followed by treatment or co-culture with parenchymal different subpopulations with various http://gut.bmj.com/ the induction of chronic with cells, demonstrating their plasticity.24 25 In sizes, a- (a-SMA) cerulein or dibutyltin dichloride, it was addition, some evidence exists that HSC expression and adhesiveness on plastic. consistently shown that BM-derived cells can also contribute to liver repair.26 With Neither method yields absolutely pure did home to the pancreas.21 22 In both regard to the pancreas, a recent study has PSC populations, although the nycodenz studies, the induction of chronic pancrea- described a subset of mitoxantrone (a type method yields a more homogeneous titis was found to be associated with an II topoisomerase inhibitor that disrupts population of PSC at the point of isolation on September 27, 2021 by guest. Protected copyright. increase in the number of GFP-expressing DNA synthesis and DNA repair in both than the outgrowth method, which is PSC. Even then, however, no more than healthy cells and cancer cells) resistant inherently prone to possible contamina- 20% of all PSC expressed GFP, indicating pancreatic cells that exhibit PSC markers, tion by other cells. In early primary a predominance of resident over BM-derived and can be induced to differentiate into cultures obtained by outgrowth methods, stellate cells.22 Together, these experimental insulin producing b cells when exposed to the contaminating cells are predominantly data point to a significant but quantita- an appropriate culture medium.27 It is and acinar cells. After tively limited contribution of BM to the possible that PSC may also express various passaging, the cultures may also contain pool of PSC. Although there are also some markers that are expressed in stem cells. a-SMA-negative fibroblasts while acinar data that EMT of acini may contribute to However, convincing functional data cells and macrophages are eliminated. the ultimate PSC population (M. Erkan showing that PSC may in fact transform Tumour cells almost never grow out from et al, unpublished data), there is general into other cell types of the pancreas are primary tumours, but may seldom grow agreementthatthereisnotcurrently not yet available. Whether PSC contribute out from metastasis samples.29 Nonethe- enough evidence to support the latter to pancreas regeneration in a manner less, good practice requires checking cell hypothesis. As of now, the situation in similar to the contribution of HSC to liver preparations for quality and purity humans is not known. The effects of BM- regeneration, by supporting the epithelial regardless of the isolation method used. derived and EMT-derived PSC on the course cells and by developing into epithelial of inflammation, repair and fibrosis of the cells,26 remains to be analysed. As of now, pancreas remains to be further elucidated.23 the situation in humans remains MARKERS OF QUIESCENT AND Regarding hepatic and pancreatic regen- completely unknown. ACTIVATED PSC eration after injury in rodents, there is When isolating primary cells from the some preliminary evidence for HSC and METHODOLOGY FOR ISOLATION OF PSC pancreas, it is of vital importance to assess PSC acting as stem cells in respective Isolation of quiescent PSC from rat the purity of the cultures not only for the organs. HSC of rats express markers such pancreas was first reported by Apte and homogeneity of PSC but also for the

2of7 Erkan M, Adler G, Apte MV, et al. Gut (2011). doi:10.1136/gutjnl-2011-301220 Leading article Gut: first published as 10.1136/gutjnl-2011-301220 on 24 November 2011. Downloaded from absence of possible contaminants. functions, eg, profibrogenic or profi- early evidence that although expressed in Another confounder is the activation of brolytic, therefore a-SMA is considered to activated PSC, Notch3, secreted frizzled PSC on plastic, which creates differences be more a transdifferentiation marker related protein 5, Wnt4 and Wnt5a are between early and late passages. There- indicative of a myofibroblast-like pheno- absent in quiescent PSC (C. Kordes, fore, several PSC selective markers have type than a marker of PSC activity unpublished data); however, there is been proposed to assess their purity and per se.30 agreement that these findings need further activity (figure 1). The most consistent With regard to other PSC markers, validation. marker of PSC quiescence is the presence researchers are in agreement that the of vitamin A droplets in the cytoplasm. expression of desmin is highly variable in PARAMETERS OF PSC ACTIVATION With regard to activated PSC, it is PSC (variance of expression also exists There is strong agreement that activation currently not clear whether these are between different species). Similarly, there of PSC in culture can be assessed by different from myofibroblasts of the is currently not enough evidence to several functional parameters. However, it pancreas and whether there are markers to conclude that nestin alone is a sufficient is also recognised that the results of differentiate these two cell types. marker to monitor the activation of PSC studies of PSC activation may be influ- However, there is strong agreement that in rodents and humans. A more enced by many different factors including a-SMA is not an exclusive marker for commonly used and reliable immunos- stellate cell purity, culture media used and activated PSC. It is also expressed by taining marker selective for rat and human age of animals harvested or (for humans) myofibroblasts, smooth muscle cells and PSC (and absent in fibroblasts) is glial donor age and disease (normal pancreas, pericytes in blood vessels as well as the fibrillar acidic protein. Some researchers , pancreatic cancer). gut wall (ie, duodenum and papilla vateri). have reported the absence of glial fibrillar While the response of PSC to individual During activation, quiescent PSC lose acidic protein expression in quiescent PSC, activating factors has been well charac- their intracellular retinoid (vitamin A) but in the opinion of experienced workers terised in vitro, researchers are also droplets (detectable by fluorescence in the field this is more likely to be due to mindful that this may not reflect the true microscopy as a characteristic blue-green differences in the quality of the antibody in-vivo situation in which: (1) PSC are autofluorescence (rapidly fading) upon used for immunostaining than a true exposed to a multiplicity of factors at any exposure to ultraviolet light at 328 nm or absence of expression of the protein. one time; (2) there is often redundancy of 350 nm) and start to express a-SMA. The The dynamic change in gene expression the effects of cytokines; (3) culture active phenotype of PSC defined by during PSC activation can be monitored conditions may influence ECM composi- a-SMA expression may have contrasting by several gene products. There is some tion; and (4) isolated PSC in culture may behave differently from those in situ where they are surrounded by other cell Stem cells Bone EMT types (figure 2). Once activated, PSC also marrow

secrete endogenous factors that can http://gut.bmj.com/ ?? further activate transformed PSC via autocrine pathways. TGFß1, TNFa, IL1, IL6, IL8 PSC express the stem/ activin-A, ox. stress (ROS) progenitor cell markers acetaldehyde, ethanol • CD34, Nestin, p75NTR, •GFAP, Bcrp1, Aldh, Notch INTRACELLULAR SIGNALLING activation - transformation PATHWAYS INVOLVED IN STELLATE CELL ACTIVATION/DEACTIVATION on September 27, 2021 by guest. Protected copyright. Quiescent PSC (fat-storing phenotype) Activated PSC ( phenotype) The intracellular pathways involved in the ‘ ’ • Localisation: periacinar and interlobular • Localisation: interlobular in fibrotic areas activation of PSC have also been inten- • Numerous perinuclear fat-droplets adjacent to carcinoma cells sively analysed as the activity of the (retinoids) • Loss of fat droplets, loss of retinoids stroma significantly impacts on the course • Express , desmin, GFAP, • α-smooth muscle actin positive nestin, • Intense rER of pancreatitis and on tumour behav- • Low mitotic index • High mitotic index iour.31 Therefore, intracellular pathways • Low capacity to synthesise ECM • Express various receptos PDGF-R, TGFß-R, ICAM-1 • High motility and contraction are considered to be important as poten- • Produce ECM proteins: Coll. I, III, XI, fibronection, tial therapeutic targets to inhibit/revert • Synthesise MMPs and TIMPs for ECM-turnover • Release neurotrophic factors/transmitters: NGF, Ach stellate cell activity. Pertinently, kinases of • Release growth factors and cytokines: PDGF, FGF, TGFß1, the mitogen-activated protein kinase CTGF, IL-1ß, IL-6, IL8, RANTES, MCP-1, ET-1, VEGF family have been implicated in many Figure 1 Activation of quiescent stellate cells. Ach, acetylcholine; Aldh, aldehyde dehydrogenase; aspects of PSC activation, but have been Bcrp1, breakpoint cluster region pseudogene 1; CD34, bluster of differentiation molecule-34; Coll, linked most consistently to the trans- collagen; CTGF, growth factor; ECM, extracellular matrix; EMT, epithelial duction of mitogenic signals and the mesenchymal transition; ET-1, endothelin-1; FGF, fibroblast-growth-factor; GFAP, glial fibrillar stimulation of cytokine/chemokine (eg, acidic protein; ICAM-1, inter-cellular adhesion molecule 1; IL1, Interleukin-1; IL-1b, interleukin-1 MCP-1 and IL-6) production.32 The latter beta; IL6, interleukin-6; IL8, interleukin-8; MCP-1, monocyte chemotactic protein 1; MMPs, matrix effect is transcriptionally mediated by AP- metalloproteinases; NGF, nerve growth factor; ox. stres, oxidative stress; p75NTR, p75 neurotrophin receptor; PDGF, platelet-derived growth factor; PDGF-R, platelet-derived growth factor 1 and nuclear factor kappa B transcription receptor; PSC, pancreatic stellate cells; RANTES, regulated upon activation, normal T-cell factors. On the other hand, Smad tran- expressed, and secreted also known as CCL5; rER, rough endoplasmic reticulum; ROS, reactive scription factors are key mediators of the oxygen species; TGFb1, transforming growth factor beta-1; TGFb-R, transforming growth factor biological effects of transforming growth beta receptor; TIMPs, tissue inhibitor of metalloproteinases; TNFa, tumor necrosis factor-alpha; factor b1 in PSC, stimulating trans- VEGF, vascular endothelial growth factor. formation to a myofibroblastic phenotype

Erkan M, Adler G, Apte MV, et al. Gut (2011). doi:10.1136/gutjnl-2011-301220 3of7 Leading article Gut: first published as 10.1136/gutjnl-2011-301220 on 24 November 2011. Downloaded from

? Bone Cancer cells marrow (CC) Neural/cancer stem cells cell Interactions: NGF, GNDF, PEDF

Cancer cells stimulate PSC: Neuropathy Proliferation (PDGF, IGF1, ET-1) Release of growth factors and Cancer cells stimulate PSC Motility (PDGF) transmitters (NGF, periostin, activation (TGFß1, TNFα and Matrix synthesis (TGFß, FGF2, sonic ACh) Hypoxia? others) hedgehog) MMP synthesis (IL1, TGFß1, TNFα, EMMPRIN)

MMPs, plasmin activation - transformation

ECM degradation: Release of growth factors (TGFß, PDGF, FGF) Quiescent PSC PSC stimulate angio- genesis in EC via: VEGF, PDGF, FGF1, IL-8 FGF2, coll-I, periostin, PSC stimulate cancer cells: Activation adrenomedullin, uPA Proliferation (TGFß, FGF2, PDGF, via hypoxia prokineticin-1 MMP-12 EGF, CTGF, AM, Gal-3, SDF-1) MMPs, uPA Chemoresistance (NO,IL-1ß, periostin) Invasion (MMPs, uPA, tPA, SDF-1) Motility (PDGF, EGF, SDF-1) CC/PSC driven anti- PSC reduce: via: MMP- Anoikis/apoptosis (coll-1, FN, periostin) driven cleavage of endostatin, angiostatin from CC. Direct EC inhibiton of CC via PEDF, TSP1 (MMPs), plasmin CC stimulated angio- genesis in EC via:

VEGF, PDGF, FGF http://gut.bmj.com/ plasmin, MMPs Endothelial cells Cancer cells

Figure 2 Interactions of pancreatic stellate cells with neighbouring cells in their microenvironment. Ach, acetylcholine; AM, adrenomedullin; CC, cancer cells; Coll, collagen; CTGF, connective tissue growth factor; EC, endothelial cells; ECM, extracellular metrix; EGF, epidermal growth factor; EMMPRIN, extracellular matrix metalloproteinase inducer also known as Basigin or CD147; ET-1, endothelin-1; FGF, fibroblast-growth-factor; GAL-3, galactoside-binding, soluble, 3; GNDF, glial cell line-derived neurotrophic factor; IGF-1, insulin-like growth factor 1; IL1, interleukin-1; IL8, interleukin-8; MMPs, matrix metalloproteinases; NGF, nerve growth factor; NO, nitric oxide; PSC, pancreatic stellate cells; PDGF, platelet-derived growth factor; PEDF, pigment derived factor; SDF-1, stromal cell-derived factor-1; TGFb1, transforming growth factor beta-1; TNFa, tumor necrosis on September 27, 2021 by guest. Protected copyright. factor-alpha; tPA, tissue plasminogen activator; TSP1, thrombospondin 1; uPA, urokinase-type plasminogen activator; VEGF, vascular endothelial growth factor. and increased ECM synthesis.32 However, albumin may also inactivate PSC.33 less adherent therefore detach earlier from while several signalling pathways medi- Therefore, modulation of PPAR-g and plastic during trypsinisation. Using this ating PSC functions have been identified, STAT1, which can be targeted by clini- difference, pure cultures of PSC and PFB it is currently not possible to clarify their cally applicable drugs (thiazolidinediones can be obtained after repeated mild tryp- ‘restricted’ role as other pathways medi- and interferons, respectively), deserves sinisation and separate cultivation.34 In ating similar functions are increasingly further investigation. contrast to PSC, which show a star-like being identified and the cross-talk shape (few are also triangular or spindle- between these pathways is also being shaped), cultured PFB are spindle-shaped elucidated. PSC ARE THE KEY FIBROGENIC CELLS and smaller. Both cell types are vimentin Signalling pathways that are respon- IN THE PANCREAS AND ARE DIFFERENT positive; 20e40% of PSC are desmin posi- sible for PSC inactivation (reversal to FROM PFB tive and more than 90% are a-SMA posi- a relatively quiescent phenotype) have Differentiation between PSC and pancre- tive (positivity increases as cells are kept also been identified in recent years. Inhi- atic fibroblasts is an important issue given on plastic).34 In contrast, PFB are desmin bition of mitogen-activated protein kinase that the outgrowth method of PSC isola- and a-SMA negative. Importantly, PSC (ERK, JNK and p38 kinase) signalling in tion involves the use of fibrotic pancreatic produce higher amounts of ECM proteins PSC mediates the induction of quiescence tissue. In contrast to pancreatic fibroblasts compared with PFB, and express the scav- in response to retinol and retinoic acid. (PFB), PSC express a-SMA and form dense enger receptor CD36, CCK receptors 1/2 There is some evidence that the forced bodies (microfilaments) like myofibroblast- and ACh receptor while PFB do not express expression of PPAR-g, C/EBP-a,or like cells.45Compared with PSC, PFB are any of these receptors.34 35

4of7 Erkan M, Adler G, Apte MV, et al. Gut (2011). doi:10.1136/gutjnl-2011-301220 Leading article Gut: first published as 10.1136/gutjnl-2011-301220 on 24 November 2011. Downloaded from VARIANCES OF PSC BETWEEN SPECIES, titis. It is proposed that the initial lesion is inhibitor of stromal angiogenesis in DONORS AND IMPACT OF autodigestive tissue necrosis, which is pancreatic cancer.49 50 However, the IMMORTALISATION ON INTERPRETING followed by inflammation and the induc- downstream targets of hedgehog signalling RESULTS tion of the fibrotic reaction.39 42 43 In stage in PSC remain to be fully elucidated. There is strong agreement that there are I with overt tissue injury, PSC are found in speciesaswellasdonor-dependentvari- close association with macrophages TRANSGENIC MOUSE MODELS OF ances between murine and human PSC. As around areas of necrosis. In stage II PANCREATIC CANCER AND RE-CREATION a result of the limited access to human with extensive cellular fibrosis, PSC are OF FIBROTIC TUMOUR pancreatic tissue at several institutions, found in the perilobular spaces. In stage III MICROENVIRONMENT immortalised PSC have been proposed as an with established dense fibrosis in the Genetically engineered mouse models of alternative model to study pancreatic perilobular spaces, PSC are conspicuously pancreatic cancer, ie, mice with a pancreas- stroma. Immortalised PSC may be useful reduced in number, and in stage IV, when specific activation of oncogenic Kras, have for studies of molecular signalling that in addition to perilobular fibrosis calculi in been instrumental in defining the steps require manipulation of gene expression the ducts are present, PSC are mainly from epithelial transformation to invasive and use in in-vivo models that require cells detected adjacent to duct ulcerations tumour growth.51 52 On a morphological to remain viable for a relatively longer time caused by calculi.43 There is general level, these models reflect the presumed period.36 37 However, it is important to note agreement that in the early stages (I and stepwise progression from intraepithelial that differences exist between immortalised II) cessation of alcohol would prevent the neoplasia to ductal adenocarcinoma. In and primary PSC, and therefore caution formation of organ fibrosis, predominantly particular, some of the mouse tumours +/Cre G12D/+ must be exercised when extrapolating through increased apoptosis of activated such as p48 ;LSL-KRAS and the e G12D/+ findings with immortalised PSC to the PSC.39 42 44 However, there is currently PDX-1-Cre;LSL-KRAS are accompa- clinical situation. Of more concern is the not enough evidence showing that nied by a significant desmoplastic observation that immortalised PSC were increasing the fibrolytic activity of PSC response, thus resembling human pancre- able (albeit occasionally) to form aggressive reverts organised fibrosis of the pancreas. atic ductal adenocarcinoma. However, it anaplastic tumours when injected alone The tumour microenvironment is has been unclear for a long time whether into nude mice (M. Buchholz et al,unpub- known to be an important contributor to a provisional matrix is already present e lished data). This raises the possibility that the malignant phenotype.36 38 41 45 48 In around precursor lesions, whether PSC are the immortalisation procedure resulted in the pancreas, PSC have been identified part of this matrix and whether desmo- the preferential selection of malignant within the tumour microenvironment of plasia is a lesion-specific response and, in subclones. Therefore, there is strong agree- pancreatic ductal adenocarcinoma. Recent particular, whether the excessive matrix ment that the results of experiments evidence from animal experiments suggests has any clinical relevance. With regard to obtained by using murine or immortalised that PSC can promote local tumour the last point, a recent paper by Erkan PSC should ideally be verified using several growth and metastatic spread and can also et al31 has shown a close correlation different primary human PSC cultures to increase resistance to chemo and radiation between the extent of a-SMA staining and http://gut.bmj.com/ e maximise the robustness of the data. treatment.36 38 41 45 48 The vascularity of poor outcome, whereas strong collagen the tumour is believed to play a role in the deposition correlated with a favourable PSC AND PANCREATIC DISEASES aggressive behaviour of pancreatic cancer. outcome in pancreatic cancer. In the Compelling evidence has accumulated in Pancreatic ductal adenocarcinoma as well genetically engineered mouse models of recent years to support a major role as chronic pancreatitis is characterised by pancreatic ductal adenocarcinoma, the forPSC in both fibrogenesis and fibrol- hypoxia and fibrosis.46 47 49 It remains earliest stromal activation and ECM e on September 27, 2021 by guest. Protected copyright. ysis.4538 41 During pancreatic injury, PSC unclear whether PSC play an overall pro- deposition are found around metaplastic/ are transformed (in response to factors angiogenic or anti-angiogenic role in dysplastic lesions such as tubular e that are now well identified, including pancreatic fibrosis and cancer.36 46 49 PSC complexes and to a lesser extent around oxidant stress, cytokines, growth factors activity is inversely correlated with PanIN lesions. However, it is not clear and toxins such as alcohol and its metab- vascular density, and inhibition of stellate whether this stromal activation is a part of olite acetaldehyde) from their quiescent cell activity results in increased vascularity carcinogenesis or an epiphenomenon state to an activated myofibroblast-like and the delivery of chemotherapeutic representing an effort to confine the phenotype that synthesises and secretes reagents.46 49 On the other hand, condi- preneoplastic lesions.53 In breast and excessive amounts of ECM proteins tioned media of PSC induce angiogenesis prostate cancers, such a stromal reaction (increased fibrogenesis). Activated PSC are both in vitro and in vivo through the has been shown to precede the actual seen in the early phases of alcoholic production of vascular endothelial growth cancer. However, despite early evidence in chronic pancreatitis and in autoimmune factor and non-vascular endothelial growth pancreatic cancer,53 judging by the stromal e pancreatitis.42 44 However, it is unclear factor family members.46 47 These contra- activation, it is currently not clear whether how fibrogenesis is initiated in the dictory findings could be explained by the tubular complexes are an additional inflamed pancreas. There are currently two dynamic rather than on/off responses of precursor lesion in pancreatic ductal concepts. The first concept focuses on the PSC. It is likely that PSC may exert adenocarcinoma. direct activation of PSC by acetaldehyde different effects on angiogenesis depending (the oxidative metabolite of alcohol) and on the site (invading front vs dense fibrotic CONCLUSIONS oxidant stress (as shown in vitro and in areas) and disease stage (early vs Pancreatic fibrosis, which develops as animal experiments).44 The second advanced). Pertinently, sonic hedgehog a consequence of chronic inflammation, concept is based on the necrosisefibrosis signalling has been implicated in PSC leads to the loss of functional parenchyma sequence as the underlying pathogenic activation and suggested as an important and probably increases the risk of cancer. mechanisms of alcoholic chronic pancrea- enhancer of the desmoplastic reaction and As the role of PSC in chronic pancreatitis

Erkan M, Adler G, Apte MV, et al. Gut (2011). doi:10.1136/gutjnl-2011-301220 5of7 Leading article Gut: first published as 10.1136/gutjnl-2011-301220 on 24 November 2011. Downloaded from and pancreatic cancer is increasingly clar- 2. Omary MB, Lugea A, Lowe AW, et al. The 22. Watanabe T, Masamune A, Kikuta K, et al. Bone ified, it is anticipated that effective pancreatic stellate cell: a star on the rise in marrow contributes to the population of pancreatic fi pancreatic diseases. J Clin Invest 2007;117:50e9. stellate cells in mice. Am J Physiol Gastrointest Liver approaches to target PSC speci cally will 3. Watari N, Hotta Y, Mabuchi Y. Morphological Physiol 2009;297:G1138e46. be developed. Such therapeutic strategies studies on a vitamin A-storing cell and its complex 23. Pinzani M. Epithelialemesenchymal transition in would be expected to reduce the fibrosis of with observed in mouse pancreatic chronic liver disease: fibrogenesis or escape from chronic pancreatitis, thereby retarding the tissues following excess vitamin A administration. death? J Hepatol 2011;55:459e65. e development of exocrine and endocrine Okajimas Folia Anat Jpn 1982;58:837 58. 24. Kordes C, Sawitza I, Haussinger D. Hepatic and fi 4. Apte MV, Haber PS, Applegate TL, et al. Periacinar pancreatic stellate cells in focus. Biol Chem insuf ciency, and interrupt the interaction stellate shaped cells in rat pancreas: identification, 2009;390:1003e12. of PSC in the stromal reaction with isolation, and culture. Gut 1998;43:128e33. 25. Sawitza I, Kordes C, Reister S, et al. The niche of pancreatic cancer cells, thereby inhibiting 5. Bachem MG, Schneider E, Gross H, et al. stellate cells within rat liver. Hepatology e tumour progression and improving the Identification, culture, and characterization of 2009;50:1617 24. pancreatic stellate cells in rats and humans. 26. Roskams T. Relationships among stellate cell otherwise dismal prognosis of this disease. Gastroenterology 1998;115:421e32. activation, progenitor cells, and hepatic regeneration. Therefore, at present, the field of PSC 6. Wake K. Perisinusoidal stellate cells (fat-storing Clin Liver Dis 2008;12:853e 60; ix. research is dynamic and wide open, with cells, interstitial cells, lipocytes), their related 27. Mato E, Lucas M, Petriz J, et al. Identification of significant potential for novel discoveries structure in and around the liver sinusoids, and a pancreatic stellate cell population with properties of and major breakthroughs that could have vitamin A-storing cells in extrahepatic organs. Int Rev progenitor cells: new role for stellate cells in the Cytol 1980;66:303e53. pancreas. Biochem J 2009;421:181e91. a lasting impact on the treatment of 7. Pinzani M, Failli P, Ruocco C, et al. Fat-storing cells 28. Vonlaufen A, Phillips PA, Yang L, et al. Isolation of patients with pancreatic diseases. The as liver-specific pericytes. Spatial dynamics of quiescent human pancreatic stellate cells: a promising Pancreatic Star Alliance would particularly agonist-stimulated intracellular calcium transients. J in vitro tool for studies of human pancreatic stellate e e like to encourage young researchers to Clin Invest 1992;90:642 6. cell biology. Pancreatology 2010;10:434 43. 8. Asahina K, Tsai SY, Li P, et al. Mesenchymal origin 29. Haag C, Stadel D, Zhou S, et al. Identification of enter this exciting area of pancreatology. of hepatic stellate cells, submesothelial cells, and c-FLIP(L) and c-FLIP(S) as critical regulators of death perivascular mesenchymal cells during mouse liver receptor-induced apoptosis in pancreatic cancer cells. FUTURE RESEARCH DIRECTIONS development. Hepatology 2009;49:998e1011. Gut 2011;60:225e37. 1. PSC origin (lineage tracing studies). 9. Enzan H, Himeno H, Hiroi M, et al. Development of 30. Krizhanovsky V, Yon M, Dickins RA, et al. 2. Functions in health. hepatic sinusoidal structure with special reference to Senescence of activated stellate cells limits liver the Ito cells. Microsc Res Tech 1997;39:336e49. fibrosis. Cell 2008;134:657e67. 3. Interactions with other pancreatic cells 10. Cassiman D, Barlow A, Vander Borght S, et al. 31. Erkan M, Michalski CW, Rieder S, et al. The activated (endocrine cells, immune cells, Hepatic stellate cells do not derive from the neural stroma index is a novel and independent prognostic Schwann cells, nerve cells). crest. J Hepatol 2006;44:1098e104. marker in pancreatic ductal adenocarcinoma. Clin e 4. Role in pancreatic repair/regeneration. 11. Kiassov AP, Van Eyken P, van Pelt JF, et al. Desmin Gastroenterol Hepatol 2008;6:1155 61. expressing nonhematopoietic liver cells during rat 32. Masamune A, Shimosegawa T. Signal transduction 5. Therapeutic targeting. liver development: an immunohistochemical and in pancreatic stellate cells. J Gastroenterol Figures presented in this article are modified from the morphometric study. Differentiation 1995;59:253e8. 2009;44:249e60. figures previously published in Cancers 2010, in which 12. Vassy J, Rigaut JP, Briane D, et al. Confocal 33. Kim N, Yoo W, Lee J, et al. Formation of vitamin A authors hold the copyright and Multidisciplinary Digital microscopy immunofluorescence localization of lipid droplets in pancreatic stellate cells requires e Publishing Institute (MDPI) is a licensee. desmin and other intermediate filament proteins in albumin. Gut 2009;58:1382 90. http://gut.bmj.com/ fetal rat . Hepatology 1993;17:293e300. 34. Bachem MG, Schmid-Kotsas A, Siech M, et al. Funding Funding of the meeting and work by the 13. Cassiman D, Roskams T. Beauty is in the eye of the Pancreatic stellate cells and their role in fibrogenesis. Pancreatic Star Alliance described in this review is beholder: emerging concepts and pitfalls in hepatic In: Johnson CD, Imrie CW, eds. Pancreatic Disease, supported by grant fundings to ME, from the DFG (GZ: stellate cell research. J Hepatol 2002;37:527e35. Basic Science and Clinical Management. London: ER 563/3-1, AOBJ: 577801), from the German Federal 14. Friedman SL. Molecular regulation of hepatic Springer-Verlag, 2004:226e39. Ministry of Education and Research (Bundesministerium fibrosis, an integrated cellular response to tissue 35. Phillips PA, Yang L, Shulkes A, et al. Pancreatic fu¨r Bildung und Forschung - BMBF -ME, MB, IE, HF, TG, injury. J Biol Chem 2000;275:2247e50. stellate cells produce acetylcholine and may play JK, CWM) within the “National Genome Research 15. Niki T, Pekny M, Hellemans K, et al. Class VI a role in pancreatic exocrine secretion. Proc Natl Network” (NGFN-Plus; 01GS08115) and Stiftung intermediate filament protein nestin is induced during Acad Sci U S A 2010;107:17397e402. on September 27, 2021 by guest. Protected copyright. Chirurgie TU Mu¨nchen. Part of the meeting costs have activation of rat hepatic stellate cells. Hepatology 36. Hwang RF, Moore T, Arumugam T, et al. Cancer- been sponsored by Abbott. 1999;29:520e7. associated stromal fibroblasts promote pancreatic Competing interests None. 16. Buchholz M, Kestler HA, Holzmann K, et al. tumor progression. Cancer Res 2008;68:918e26. Transcriptome analysis of human hepatic and 37. Jaster R, Lichte P, Fitzner B, et al. Peroxisome Contributors ME appears as the corresponding author. pancreatic stellate cells: organ-specific variations of proliferator-activated receptor gamma overexpression All authors have contibuted equally to the manuscript. a common transcriptional phenotype. J Mol Med inhibits pro-fibrogenic activities of immortalised rat e Provenance and peer review Commissioned; 2005;83:795 805. pancreatic stellate cells. J Cell Mol Med e externally peer reviewed. 17. Erkan M, Weis N, Pan Z, et al. Organ-, inflammation- 2005;9:670 82. and cancer specific transcriptional fingerprints of 38. Bachem MG, Schunemann M, Ramadani M, et al. Revised 24 October 2011 pancreatic and hepatic stellate cells. Mol Cancer Pancreatic carcinoma cells induce fibrosis by Accepted 25 October 2011 2010;9:88. stimulating proliferation and matrix synthesis of 18. Asahina K, Zhou B, Pu WT, et al. Septum stellate cells. Gastroenterology 2005;128:907e21. Gut 2011;-:1e7. doi:10.1136/gutjnl-2011-301220 transversum-derived mesothelium gives rise to 39. Kloppel G, Detlefsen S, Feyerabend B. Fibrosis of the hepatic stellate cells and perivascular mesenchymal pancreas: the initial tissue damage and the resulting cells in developing mouse liver. Hepatology pattern. Virchows Arch 2004;445:1e8. 2011;53:983e95. 40. Phillips PA, McCarroll JA, Park S, et al. Rat 19. Higashiyama R, Moro T, Nakao S, et al. Negligible pancreatic stellate cells secrete matrix contribution of bone marrow-derived cells to collagen metalloproteinases: implications for extracellular This paper is freely available online under the BMJ production during hepatic fibrogenesis in mice. matrix turnover. Gut 2003;52:275e82. Journals unlocked scheme, see http://gut.bmj.com/site/ Gastroenterology 2009;137:1459e66 e1. 41. Vonlaufen A, Joshi S, Qu C, et al. Pancreatic stellate about/unlocked.xhtml. 20. Russo FP, Alison MR, Bigger BW, et al. The bone cells: partners in crime with pancreatic cancer cells. marrow functionally contributes to liver fibrosis. Cancer Res 2008;68:2085e93. REFERENCES Gastroenterology 2006;130:1807e21. 42. Ammann RW, Heitz PU, Kloppel G. Course of 1. Friedman SL. Hepatic stellate cells: protean, 21. Sparmann G, Kruse ML, Hofmeister-Mielke N, et al. alcoholic chronic pancreatitis: a prospective multifunctional, and enigmatic cells of the liver. Bone marrow-derived pancreatic stellate cells in rats. clinicomorphological long-term study. Physiol Rev 2008;88:125e72. Cell Res 2010;20:288e98. Gastroenterology 1996;111:224e31.

6of7 Erkan M, Adler G, Apte MV, et al. Gut (2011). doi:10.1136/gutjnl-2011-301220 Leading article Gut: first published as 10.1136/gutjnl-2011-301220 on 24 November 2011. Downloaded from 43. Detlefsen S, Sipos B, Feyerabend B, et al. hypoxia-fibrosis cycle in pancreatic ductal 50. Bailey JM, Swanson BJ, Hamada T, et al. Sonic Fibrogenesis in alcoholic chronic pancreatitis: the role adenocarcinoma. Neoplasia 2009;11:497e508. hedgehog promotes in pancreatic of tissue necrosis, macrophages, myofibroblasts and 47. Masamune A, Kikuta K, Watanabe T, et al. cancer. Clin Cancer Res 2008;14:5995e6004. cytokines. Mod Pathol 2006;19:1019e26. Hypoxia stimulates pancreatic stellate cells to induce 51. Hingorani SR, Petricoin EF, Maitra A, et al. 44. Vonlaufen A, Phillips PA, Xu Z, et al. Withdrawal of fibrosis and angiogenesis in pancreatic cancer. Am J Preinvasive and invasive ductal pancreatic cancer and alcohol promotes regression while continued alcohol Physiol Gastrointest Liver Physiol 2008;295: its early detection in the mouse. Cancer Cell intake promotes persistence of LPS-induced pancreatic G709e17. 2003;4:437e50. injury in alcohol-fed rats. Gut 2011;60:238e46. 48. Xu Z, Vonlaufen A, Phillips PA, et al. Role of 52. Ji B, Tsou L, Wang H, et al. Ras activity levels control 45. Erkan M, Kleeff J, Gorbachevski A, et al. Periostin pancreatic stellate cells in pancreatic cancer the development of pancreatic diseases. creates a tumor-supportive microenvironment in the metastasis. Am J Pathol 2010;177:2585e96. Gastroenterology 2009;137:1072e82; 82 e1e6. pancreas by sustaining fibrogenic stellate cell activity. 49. Olive KP, Jacobetz MA, Davidson CJ, et al. Inhibition 53. Esposito I, Seiler C, Bergmann F, et al. Hypothetical Gastroenterology 2007;132:1447e64. of Hedgehog signaling enhances delivery of progression model of pancreatic cancer with origin in 46. Erkan M, Reiser-Erkan C, Michalski CW, et al. chemotherapy in a mouse model of pancreatic the centroacinar-acinar compartment. Pancreas Cancerestellate cell interactions perpetuate the cancer. Science 2009;324:1457e61. 2007;35:212e17.

PAGE fraction trail=6.25 http://gut.bmj.com/ on September 27, 2021 by guest. Protected copyright.

Erkan M, Adler G, Apte MV, et al. Gut (2011). doi:10.1136/gutjnl-2011-301220 7of7