Introduction Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix proteins. Pancreatic stellate cells (PSC) have the capacity to synthesise both matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinase (TIMPs). These may play a key role in pancreatic fibrogenesis by protecting extracellular matrix from degradation and supporting the survival of fibrogenic PSC. Therefore it was hypothesised that TIMP-1 has proproliferative and anti-apoptotic effects on culture activated PSC. Methods Culture activated rat PSC were used between passages 2 and 4 and serum deprived prior to all experiments. Apoptosis was induced with cycloheximide (50 mm/l) in the presence or absence of recombinant rat TIMP-1 (1–200 ng/ml). Apoptosis was quantified using acridine orange and counting of apoptotic bodies. PSC proliferation was determined by [3H]-thymidine incorporation. Culture-activated rat PSC were transfected with TIMP-1 and a negative control siRNA by electroporation. Cytotoxicity of siRNA was assessed by lactate dehydrogenase activity assay. Culture supernatants were collected to quantitate TIMP-1 protein knockdown following electroporation. Results TIMP-1 showed a dose dependent inhibition of cycloheximide induced PSC apoptosis. TIMP-1 at 100 ng/ml and 200 ng/ml significantly reduced apoptosis by up to 61% at 6 h, and up to 74% at 24 h (see table). Although there was a trend towards TIMP-1 stimulation of increased proliferation, this did not reach statistical significance. TIMP-1 electroporation was an effective means of silencing PSC TIMP-1 expression. Peak knockdown of target TIMP-1 protein was 77% at 24 h for TIMP-1 compared to negative control siRNA (p<0.05). There was no increase in cell toxicity in TIMP-1 siRNA treated cells compared with electroporation alone. Conclusion These data suggest that TIMP-1 reduces PSC apoptosis in the presence of cycloheximide, with no significant effect on proliferation. These data also suggest that siRNA are an effective means of silencing TIMP-1 in PSC. This may represent an important therapeutic target in the treatment of pancreatic fibrosis.

Introduction Pancreatic stellate cells (PSC) are exposed to supranormal concentrations of insulin via the islet–acinar portal system. We initially hypothesised that this might influence PSC numbers by effects on proliferation or apoptosis. This effect could be mediated via insulin receptors or via receptors for insulin-like growth factor (IGF-1). In this context, we examined the role of IGF-1 and insulin in the prevention of pancreatic stellate cell apoptosis. Following initial experiments, it became apparent that PSC can synthesise IGF-1, we therefore examined whether IGF-1 might be an autocrine growth factor in cultured stellate cells. The aims were to determine (1) the expression of IGF-1 and insulin receptor by PSC; (2) the secretion of IGF-1 by pancreatic stellate cells; (3) the effects of IGF-1 on PSC proliferation and apoptosis. Methods Culture-activated rat PSCs were used between passages 1 and 4 and serum deprived prior to all experiments. PSC proliferation was determined by [3H]-thymidine incorporation. Apoptosis was determined with acridine orange counting of apoptotic bodies and caspase 3 activity assays. Western blotting and immunocytochemistry were used to demonstrate the presence of IGF-1 and insulin receptors. Real-time quantitative PCR was used to detect receptor mRNA. A sandwich ELISA was used to determine the secretion of IGF-1, and real-time quantitative PCR use to confirm mRNA expression. Results Activated PSC express protein and mRNA for IGF-1 and insulin receptors. IGF-1 secretion is upregulated 2.40-fold following serum withdrawal compared with 16% FCS (see Abstract 001). This upregulation is also seen at the level of mRNA in response to serum withdrawal. Exogenous IGF-1 (100 ng/ml) and insulin (10 000 ng/ml) prevented cycloheximide-induced PSC apoptosis in a concentration-dependent manner, 63% at 24 h, and 71% at 24 h, respectively. There was moderate effect on PSC proliferation following exposure to IGF-1 but not with insulin. Conclusion These data suggest that IGF-1 has an autocrine role in reducing PSC apoptosis on serum withdrawal. Inhibiting IGF-1 expression or blocking the IGF-1 receptor may increase susceptibility to apoptosis. This represents a potential therapeutic target for prevention or treatment of pancreatic fibrosis.

Introduction: In chronic pancreatitis, pancreatic stellate cells (PSCs) become activated to a myofibroblastic phenotype secreting abundant fibrillar collagen leading to pancreatic fibrosis. In injured liver, the resolution of fibrosis requires the apoptosis of activated stellate cells. Therefore, factors which inhibit PSC apoptosis might promote pancreatic fibrosis. Regulation of PSC apoptosis is poorly understood. Two closely related polypeptide hormones, IGF-1 and insulin prevent apoptosis of various cells including hepatic stellate cells. PSCs are exposed to high levels of insulin in the insulo-acinar portal system. We have examined the role of IGF-1 and insulin on PSC survival which may be of significance in the resolution of pancreatic fibrosis. Method: Rat PSCs were used between passages 2-4 and serum deprived prior to all experiments. PSC were then treated with IGF-1(1-100ng/ml) and insulin (1-10000ng/ml) and apoptosis was induced with 50μM cycloheximide. PSC apoptosis was quantified using acridine orange and counting of apoptotic bodies. Caspase-3 activity was quantified using a commercially available kit. Results: Both insulin and IGF-1 acted as survival factors for cycloheximide induced apoptosis. In acridine orange studies, relative to cycloheximide induced apoptosis, IGF-1, 100ng/ml reduced apoptosis by 63% at 6 hrs and 24 hrs. Insulin, 10000ng/ml reduced apoptosis by 81% at 6 hrs and 71% at 24 hrs (p < 0.05, n = 4). There was a concentration dependent effect in both groups. Similar results were also observed in experiments measuring Caspase-3 activity. After 6 hrs incubation with IGF-1, 100ng/ml there was a 56% reduction in caspase activity compared with cycloheximide alone (p < 0.05, n = 3). For insulin 1000ng/ml there was a 61% reduction in caspase activity at 6 hrs. Conclusion: Our preliminary findings demonstrate that both insulin and IGF-1 prevent PSC apoptosis in the presence of cycloheximide. These molecules might therefore promote fibrosis following pancreatic injury. The uniquely high insulin concentrations adjacent to the islets might further favor the persistence of activated PSCs.

This short review examines two examples of studies into the mechanisms of allergic responses which have particular relevance to inflammation research. The first is the ability of human skin mast cells, but not those derived from lung, adenoids, tonsils or intestine, to release histamine in response to stimulation by neuropeptides including substance P, vasoactive intestinal polypeptide (VIP) and somatostatin. The neuropeptide activation site does not appear to be a classical tachykinin receptor but rather a binding site of low affinity and low specificity capable of interacting with neuropeptides and compounds with similar physicochemical characteristics. In contrast to IgE-dependent activation, neuropeptide stimulation of skin mast cells induces a rapid release of histamine with minimal generation of PGD2 and LTC4. This pseudo-allergic reaction is thought to underlie the weal and flare response in the skin and may have a role in urticaria. The second example describes studies to elucidate the mechanisms of the late asthmatic response by use of a guinea-pig model. As in man, both early and late phase responses in the guinea-pig are inhibited by sodium cromoglycate whereas only the early response is inhibited by theβ-adrenoceptor stimulant drug salbutamol. Examination of bronchoalveolar fluid has shown a temporal relationship between an airways neutrophilia and the late response. However, pharmacological manipulation and the use of an anti-neutrophil serum has shown that these events are not interdependent. The role of the airways eosinophilia requires further investigation.

PURPOSE:The purpose of this study was to determine the role of functional interactions between pancreatic cancer cells and pancreatic stellate cells (PSCs) in the formation of the desmoplastic reaction (DR) in pancreatic cancer and to characterize the effect of type I collagen (the predominant component of the DR) on pancreatic cancer cell phenotype.EXPERIMENTAL DESIGN:PSCs and type I collagen were identified in sections of pancreatic cancer using immunohistochemistry, and their anatomic relationship was studied. Interactions among pancreatic cancer cell lines (MIA PaCa-2, Panc-1, and AsPC-1), primary cultures of human PSCs, and type I collagen were investigated in a series of tissue culture models.RESULTS:In vivo, the DR causes gross distortion of normal pancreas, bringing cancer cells into close contact with numerous PSCs and abundant type I collagen. In tissue culture models of pancreatic cancer, conditioned media from each cell line increased PSC [3H]thymidine incorporation up to 6.3-fold that of controls, and AsPC-1 cells also increased PSC collagen synthesis 1.3-fold. Type I collagen was observed to increase long-term survival of pancreatic cancer cells treated with 5-fluorouracil, by up to 62% in clonogenic assays. This was because type I collagen increased the proliferation of cancer cells ([3H]thymidine incorporation was up to 2.8-fold that of cells cultured on tissue culture plastic) and reduced apoptosis of AsPC-1 cells in response to 5-fluorouracil (by regulating mcl-1).CONCLUSIONS:These experiments elucidate a mechanism by which the DR in pancreatic cancer may form and, via the collagen within it, promote the malignant phenotype of pancreatic cancer cells, suggesting significant detriment to the host.

Transforming growth factor beta-1 (TGF-β1) plays a pivotal role in tissue fibrogenesis. Understanding the factors that control resolution of fibrosis is critical to devising means to combat clinical fibrosis. Future challenges would include designing ways to block the fibrosis-specific actions of TGF-β. Blockade of transforming growth factor beta (TGF-β) activity in vivo in animal models has proven to be an effective means of inhibiting the fibrotic response to injury in various organs. Similarly, transgenic animals in which TGF-β1 expression is artificially enhanced show marked spontaneous fibrosis or increased fibrotic response to injury. TGF-β is known to effect fibroplasias, not only by its well known action of increasing extracellular matrix synthesis but also by coordinately regulating key proteins which mediate connective tissue homeostasis. This includes down-regulation of interstitial collagenase and other matrix metalloproteinases and up-regulation of antiproteases such as tissue inhibitor of metalloproteinase I and plasminogen activator inhibitor. Whilst inhibition of TGF-β activity appears to be well tolerated in rodents over several weeks, the ultimately lethal phenotype of TGF-β1 knockout mice warns us that this pluripotent cytokine is essential for normal health. Therefore, downstream pathways activated by TGF-β, which might be specific for its fibrotic effects, might be more useful targets for human fibrotic disease therapy. For example, the TGF-β response protein connective tissue growth factor may be a good target for antifibrotics but definitive evidence awaits development of suitable genetically modified animal models and specific inhibitors.

Background/Aims: Resolution of liver fibrosis is possible but the identity of the matrix metalloproteinases (MMPs) which degrade the accumulated collagens is uncertain. We examined MMP-2 and MMP-14 expression in established and resolving fibrosis to assess their role in resolution of liver fibrosis. Methods: MMP and tissue inhibitor of metalloproteinase (TIMP)-2 expression in liver extracts was examined by ribonuclease protection assay, Western blotting and gelatin zymography. MMP activity was examined by C-14 gelatin degradation. Results: In human cirrhotic liver, MMP-14 mRNA was increased to 230-330% of normal liver expression. Both 63 kDa proenzyme and 60 kDa activated form were present. Cirrhotic livers had 270-320% of normal liver expression of MMP-2 protein with 20-25% being the 62 Da activated form. Protein and mRNA for MMP-2 and MMP-14 progressively increased during 8 weeks of CCl4 treatment in rats. Between 3 and 7 days of resolution from CCl4 liver fibrosis, MMP-2 and MMP-14 persisted at elevated levels. Gelatinolytic activity in liver homogenates peaked at 7 days of recovery, being 140% above that in livers at peak fibrosis. Conclusions: Increased expression and activation of MMP-2 and -14 occurs even under conditions of elevated TIMPs during liver fibrogenesis. During liver fibrosis resolution, as TIMP expression decays, the persistence of MMP-2 and MMP-14 may permit collagen degradation.

Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possibility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of alpha(v)beta(3) integrin. alpha(v)beta(3) integrin was expressed by activated, myofibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence alpha(v) subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These alpha(v)beta(3) antagonists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by activated stellate cells was reduced by the alpha(v)beta(3) antagonists, while matrix metalloproteinase-9 synthesis was enhanced. Stellate cells incubated with active recombinant matrix metalloproteinase-9 showed enhanced apoptosis, while cells treated with a synthetic inhibitor of this protease showed increased survival. Our studies suggest that alpha(v)beta(3) integrin regulates the fate of hepatic stellate cells. Degradation of alpha(v)beta(3) ligands surrounding activated stellate cells during resolution of liver fibrosis might decrease alpha(v)beta(3) integrin ligation, suppressing stellate cell proliferation and inducing a fibrolytic, matrix metalloproteinase-secreting phenotype that may prime stellate cells for apoptosis.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.

There has long been conjecture over the degree to which there may be structural and functional heterogeneity in the tetrameric serine protease tryptase (EC 3.4.21.59), a major mediator of allergic inflammation. We have applied 2D gel electrophoresis to analyze the extent, nature, and variability of this heterogeneity in lysates of mast cells isolated from lung and skin, and in preparations of purified tryptase. Gels were silver stained, or the proteins transferred to nitrocellulose blots and probed with either tryptase-specific monoclonal antibodies or various lectins. Tryptase was the major protein constituent in mast cell lysates, and presented as an array of 9-12 diffuse immunoreactive spots with molecular masses ranging from 29 to 40 kDa, and pI values from 5.1 to 6.3. Although the patterns obtained for lung and skin tryptase were broadly similar, differences were observed between tissues and between individual donors. Lectin binding studies indicated the presence of mono-antennary or bi-antennary complex-type oligosaccharide with varying degrees of sialylation. Deglycosylation with protein-N-glycosidase F (PNGase F) reduced the size of both lung and skin tryptase, while incubation with PNGase F or neuraminidase narrowed the pI range, indicating variable degrees of glycosylation as a major contributor to the size and charge heterogeneity. Comparison of different purified preparations of lung and skin tryptase revealed no significant difference in pH profiles, but differences were seen in reactivity towards a range of chromogenic substrates, with substantial differences in Km, kcat and degree of cooperativity. Mathematical modeling indicated that the variety in kinetics parameters could not result solely from the sum of varying amounts of isoforms obeying Michaelis-Menten kinetics but with different values of Km and kcat. The heterogeneity demonstrated for tryptase in these studies suggests that there are important differences in tryptase function in different tissues.

A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.