Brit Fitzner

Peroxisome proliferator - activated receptor γ overexpression inhibits pro-fibrogenic activities of immortalised rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. In response to pro‐fibrogenic mediators, PSCs undergo an activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts. Ligands of the peroxisome proliferator‐activated receptor gamma (PPARγ), such as thiazolidinediones, are potent inhibitors of stellate cell activation and fibrogenesis in pancreas and liver. The effects of PPARγ ligands, however, however, are at least in part mediated through PPARγ‐independent pathways. Here, we have chosen a different approach to study regulatory functions of PPARγ in PSCs. Using immortalised rat PSCs, we have established a model of tetracycline (tet)‐regulated PPARγ over‐expression. Induction of PPARγ expression strongly inhibited proliferation and enhanced the rate of apoptotic cell death. Furthermore, PPARγ‐overexpressing cells synthesised less collagen than controls. To monitor effects of PPARγ on PSC gene expression, we employed Affymetrix microarray technology. Using stringent selection criteria, we identified 21 up‐ and 19 down‐regualated genes in PPARγ‐overexpressing cells. Most of the corresponding gene products are either involved in lipid metabolism, play a role in signal transduction, or are secreted molecules that regulate cell growth and differentition. In conclusion, our data suggest an active role of PPARγ in the induction of a quiescent PSC phenotype. PPARγ‐regulated genes in PSCs may serve as novel targets for the development of antifibrotic therapies.

Regulation of pancreatic stellate cell function in vitro: biological and molecular effects of all-trans retinoic acid

Pancreatic stellate cells (PSCs) are essentially involved in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. Profibrogenic mediators, such as ethanol metabolites and cytokines, induce a PSC activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts which includes a loss of the characteristic retinoid-containing fat droplets. Here, we have analysed how exogenous all-trans retinoic acid (ATRA) affects activation of rat PSCs induced by sustained culture. Bromodeoxyuridine-incorporation assays indicated an ATRA-dependent inhibition of DNA synthesis. In contrast, ATRA did not affect expression of alpha-smooth muscle actin, a protein typical for myofibroblasts. Quantification of [3H]proline incorporation revealed a diminished collagen production in ATRA-treated PSCs. Furthermore, zymography experiments showed that supernatants of ATRA-exposed PSC cultures contained higher levels of matrix metalloproteinase-9 but not of matrix metalloproteinase-2 than untreated controls. At the level of intracellular signalling, ATRA had no effect on extracellular signal-regulated kinase activation after incubation of PSCs with the mitogen platelet-derived growth factor (PDGF). In addition, PDGF-induced DNA binding of activator protein-1 (AP-1) transcription factors was not inhibited by ATRA treatment. Luciferase reporter gene assays, however, revealed an ATRA-dependent transrepression of AP-1 in PDGF-stimulated PSCs. Together, the results indicate that exogenous ATRA displays inhibitory effects on PSC proliferation and collagen synthesis but does not block phenotypic transition towards myofibroblasts. We hypothesise that inhibition of AP-1 signalling may be involved in the mediation of biological effects of ATRA on PSCs.

Molecular insights into connective tissue growth factor action in rat pancreatic stellate cells.

Pancreatic fibrosis, a key feature of chronic pancreatitis and pancreatic cancer, is mediated by activated pancreatic stellate cells (PSC). Connective tissue growth factor (CTGF) has been suggested to play a major role in fibrogenesis by enhancing PSC activation after binding to alpha5beta1 integrin. Here, we have focussed on molecular determinants of CTGF action. Inhibition of CTGF expression in PSC by siRNA was associated with decreased proliferation, while application of exogenous CTGF stimulated both cell growth and collagen synthesis. Real-time PCR studies revealed that CTGF target genes in PSC not only include mediators of matrix remodelling but also the proinflammatory cytokines interleukin (IL)-1beta and IL-6. CTGF stimulated binding of NF-kappaB to the IL-6 promoter, and siRNA targeting the NF-kappaB subunit RelA interfered with CTGF-induced IL-6 expression, implicating the NF-kappaB pathway in the mediation of the CTGF effect. In further studies, we have analyzed regulation of CTGF expression in PSC. Transforming growth factor-beta1, activin A and tumor necrosis factor-alpha enhanced expression of the CTGF gene, while interferon-gamma displayed the opposite effect. The region from -74 to -125 of the CTGF promoter was revealed to be critical for its activity in PSC as well as for the inhibitory effect of interferon-gamma. Taken together, our results indicate a tight control of CTGF expression in PSC at the transcriptional level. CTGF promotes fibrogenesis both directly by enhancing PSC proliferation and matrix protein synthesis, and indirectly through the release of proinflammatory cytokines that may accelerate the process of chronic inflammation.

Transforming growth factor-beta induces nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway.

Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis. Transforming growth factor (TGF)-β, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75NTR expression was weak in prPSC. In contrast to ihPSC TGF-β activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1. We conclude that under conditions of upregulated TGF-β, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.

Senescence determines the fate of activated rat pancreatic stellate cells

In chronic pancreatitis (CP), persistent activation of pancreatic stellate cells (PSC) converts wound healing into a pathological process resulting in organ fibrosis. Here, we have analysed senescence as a novel mechanism involved in the termination of PSC activation and tissue repair. PSC senescence was first studied in vitro by establishing long‐term cultures and by applying chemical triggers, using senescence‐associated β‐Galactosidase (SA β‐Gal) as a surrogate marker. Subsequently, susceptibility of PSC to immune cell‐mediated cytolysis was investigated employing cocultures. Using the model of dibutyltin dichloride‐induced CP in rats, appearance of senescent cells was monitored by immunohistochemistry and immunofluorescence, and correlated with the progression of tissue damage and repair, immune cell infiltration and fibrosis. The results indicated that long‐term culture and exposure of PSC to stressors (doxorubicin, H2O2 and staurosporine) induced senescence. Senescent PSC highly expressed CDKN1A/p21, mdm2 and interleukin (IL)‐6, but displayed low levels of α‐smooth muscle actin. Senescence increased the susceptibility of PSC to cytolysis. In CP, the number of senescent cells correlated with the severity of inflammation and the extension of fibrosis. Areas staining positive for SA β‐Gal overlapped with regions of fibrosis and dense infiltrates of immune cells. Furthermore, a close physical proximity of immune cells and activated PSC was observed. We conclude that inflammation, PSC activation and cellular senescence are timely coupled processes which take place in the same microenvironment of the inflamed pancreas. Lymphocytes may play a dual‐specific role in pancreatic fibrogenesis, triggering both the initiation of wound healing by activating PSC, and its completion by killing senescent stellate cells.

Mathematical modelling of interferon-γ signalling in pancreatic stellate cells reflects and predicts the dynamics of STAT1 pathway activity

Signal transducer and activator of transcription (STAT) 1 is essentially involved in the mediation of antifibrotic interferon-gamma (IFN gamma) effects in pancreatic stellate cells (PSC). Here, we have further analysed the activation of the STAT1 pathway in a PSC line by combining quantitative data generation with mathematical modelling. At saturating concentrations of IFN gamma, a triphasic pattern of STAT1 activation was observed. An initial, rapid induction of phospho-STAT1 was followed by a plateau phase and another, long-lasting phase of further increase. The late increase occurred despite enhanced expression of the feedback inhibitor (SOCS1), and corresponded to increased levels of total STAT1 protein. If IFN gamma was applied at non-saturating concentrations, phospho-STAT1 and SOCS1 levels peaked and declined again over a 12 hour period, while STAT1 protein levels remained high. The mathematical model, based on a system of ordinary differential equations, describes temporal changes of the network components as a function of interactions and transport processes. The model reproduced activation profiles of all components of the STAT1 pathway that were experimentally analysed. Furthermore, it successfully predicted the dynamics of network components in additional experimental studies. Based on experimental findings and the results obtained from modelling, we suggest exhaustion of applied IFN gamma and STAT1 dephosphorylation by tyrosine phosphatases as limiting factors of STAT1 activation in PSC. In contrast, we did not obtain compelling evidence that SOCS1 acts as an efficient feedback inhibitor in our experimental system. We believe that further investigations into mathematical modelling of the STAT1 pathway will improve the understanding of the antifibrotic interferon action.

Molecular biomarkers in cerebrospinal fluid of multiple sclerosis patients

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system, usually occurring in young adults and leading to disability. Despite the progress in technology and intensive research work of the last years, diagnosing MS can still be challenging. A heterogenic and complex pathophysiology with various types of disease courses makes MS unique for each patient. There is an urgent need to identify markers facilitating rapid and accurate diagnosis and prognostic assessments with regard to optimal therapy for each MS patient. Cerebrospinal fluid (CSF) is an outstanding source of specific markers related to MS pathology. Molecules reflecting specific pathological processes, such as inflammation, cellular damage, and loss of blood-brain-barrier integrity, are detectable in CSF. Clinically used biomarkers of CSF are oligoclonal bands, IgG-index, measles-rubella-zoster-reaction, anti-aquaporin 4 antibodies, and antibodies against John Cunningham virus. Many other potential biomarkers have been proposed in recent years. In this review we examine the current scientific knowledge on CSF molecular markers that could guide diagnosis and discrimination of different MS forms, support treatment decisions, or be helpful in monitoring and predicting disease progression, therapy response, and complications such as opportunistic infections.

Studies on mechanisms of interferon-gamma action in pancreatic cancer using a data-driven and model-based approach

BackgroundInterferon-gamma (IFNγ) is a multifunctional cytokine with antifibrotic and antiproliferative efficiency. We previously found that pancreatic stellate cells (PSC), the main effector cells in cancer-associated fibrosis, are targets of IFNγ action in the pancreas. Applying a combined experimental and computational approach, we have demonstrated a pivotal role of STAT1 in IFNγ signaling in PSC. Using in vivo and in vitro models of pancreatic cancer, we have now studied IFNγ effects on the tumor cells themselves. We hypothesize that IFNγ inhibits tumor progression through two mechanisms, reduction of fibrogenesis and antiproliferative effects on the tumor cells. To elucidate the molecular action of IFNγ, we have established a mathematical model of STAT1 activation and combined experimental studies with computer simulations.ResultsIn BALB/c-nu/nu mice, flank tumors composed of DSL-6A/C1 pancreatic cancer cells and PSC grew faster than pure DSL-6A/C1 cell tumors. IFNγ inhibited the growth of both types of tumors to a similar degree. Since the stroma reaction typically reduces the efficiency of therapeutic agents, these data suggested that IFNγ may retain its antitumor efficiency in PSC-containing tumors by targeting the stellate cells. Studies with cocultures of DSL-6A/C1 cells and PSC revealed a modest antiproliferative effect of IFNγ under serum-free conditions. Immunoblot analysis of STAT1 phosphorylation and confocal microscopy studies on the nuclear translocation of STAT1 in DSL-6A/C1 cells suggested that IFNγ-induced activation of the transcription factor was weaker than in PSC. The mathematical model not only reproduced the experimental data, but also underscored the conclusions drawn from the experiments by indicating that a maximum of 1/500 of total STAT1 is located as phosphorylated STAT1 in the nucleus upon IFNγ treatment of the tumor cells.ConclusionsIFNγ is equally effective in DSL-6A/C1 tumors with and without stellate cells. While its action in the presence of PSC may be explained by inhibition of fibrogenesis, its efficiency in PSC-free tumors is unlikely to be caused by direct effects on the tumor cells alone but may involve inhibitory effects on local stroma cells as well. To gain further insights, we also plan to apply computer simulations to the analysis of tumor growth in vivo.

Identification of quantitative trait loci for murine autoimmune pancreatitis

Background and aims Autoimmune pancreatitis (AIP) represents a rare but clinically relevant cause of pancreatic inflammation. Using MRL/Mp mice as a model of spontaneous AIP, the genetic basis of the disease was studied. Methods To identify quantitative trait loci (QTL) of AIP, an advanced intercross line was studied, originating from MRL/MpJ parental mice and the following three mouse strains: Cast (healthy controls), BXD2 (susceptible to collagen induced arthritis), and NZM (a model of lupus erythematosus). This concept was chosen to identify both general autoimmune disease associated loci and AIP specific QTL. Therefore, generation G4 of outbred intercross mice was characterised phenotypically by scoring histopathological changes of the pancreas and genotyped with single nucleotide polymorphism (SNP) arrays. Data were analysed with the R implementation of HAPPY. Results Five QTLs, correlating with the severity of AIP, were identified. Two of them mapped to chromosome 4 and one to chromosomes 2, 5, and 6, respectively. The QTL on chromosome 6 displays the highest LOD score (5.4) and contains the C-type lectin domain family 4 member a2 in its peak region, which encodes a receptor protein of dendritic cells that has previously been implicated in autoimmune diseases such as Sjogrens syndrome. AIP candidate genes of other QTLs include heterogeneous nuclear ribonucleoprotein A3; nuclear factor, erythroid derived 2, like 2; Sjogren syndrome antigen B; and ubiquitin protein ligase E3 component n-recognin 3. Conclusions This study has identified QTLs and putative candidate genes of murine AIP. Their functional role and relevance to human AIP will be studied further.

Synergistic growth inhibitory effects of the dual endothelin-1 receptor antagonist bosentan on pancreatic stellate and cancer cells.

Pancreatic stellate cells (PSC) play a key role in pancreatic fibrosis. Activation of PSC occurs in response to pro-fibrogenic stimuli and is maintained by autocrine loops of mediators, such as endothelin (ET)-1. Here, we have evaluated effects of the dual ET receptor antagonist bosentan in models of pancreatic fibrogenesis and cancer. Cell culture studies revealed that PSC and DSL6A pancreatic cancer cells expressed both ET-1 and ET receptors. Bosentan efficiently inhibited proliferation of both cell types and collagen synthesis in PSC. Expression of the myofibroblastic marker α-smooth muscle actin, connective tissue growth factor, and ET-1 itself in PSC was reduced, while expression of matrix metalloproteinase-9 was enhanced. Like PSC, DSL6A cells secrete less ET-1 when cultured with bosentan. In a rat model of pancreatic fibrosis, chronic pancreatitis induced by dibutyltin dichloride, a tendency towards a diminished disease progression was observed in a subgroup of rats with less severe disease. Together, our results indicate that bosentan exerts antifibrotic and antitumor effects in vitro. Its efficiency in vivo warrants further investigation.