Both chronic pancreatitis and pancreatic cancer are accompanied by an organ fibrosis 12. The progressive replacement of pancreas-specific tissue by ECM-rich connective tissue leads to the development of an exocrine and endocrine insufficiency of the gland. So far, specific therapies to prevent, retard or even reverse this process are not available.

Fibroblast activation has been reported to be a common event in pancreatitis already more than a decade ago 789, but the basic matrix producing cell type in the pancreas remained to be identified. In 1997, Saotome et al. 10 described the isolation of periacinar fibroblast-like cells from human pancreas. The cells displayed some characteristics of activated myofibroblasts, e.g. expression of α-smooth muscle actin (α-SMA) and synthesis of ECM proteins. One year later, Bachem et al. 3 and Apte et al. 4 found that vitamin A-storing cells resembling hepatic stellate cells can be isolated from human and rat pancreas. In the healthy organ, PSCs comprise about 4% of all pancreatic cells and show a periacinar distribution. They can be identified by the presence of retinoid-containing cytoplasmic lipid droplets and by immunostaining for cytoskeletal proteins such as desmin and glial fibrillary acidic protein 4. In culture, pancreatic stellate cells readily grow 4 and change from a quiescent phenotype to a myofibroblast-like cell expressing α-SMA and producing large amounts of the ECM proteins collagen type I and III, fibronectin as well as laminin 3. This activation process is accompanied by a loss of the characteristic retinoid-containing fat droplets 34. Together, these in vitro data gave rise to the hypothesis that PSCs might play a pivotal role in pancreatic fibrogenesis.

In the meantime, this hypothesis has been supported by the results of several in vivo studies using experimental models of pancreatic fibrosis: Infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct of rats causes a pancreatic necroinflammation followed by fibrosis 11. In TNBS-treated rats, areas of pancreatic fibrosis colocalized with α-SMA-positive cells, suggesting the presence of activated PSCs. Furthermore, dual staining techniques indicated that these α-SMA-positive cells were the main source of collagen in the fibrotic pancreas 12. Importantly, very similar data were obtained when pancreatic tissue from patients with chronic pancreatitis was analyzed 12. Another well-established model of fibrogenesis involves the administration of a single intravenous dose of dibutyltin dichloride (8 mg/kg body weight), resulting in the development of a chronic pancreatitis associated with fibrosis 13. Time course studies of DBTC-induced chronic pancreatitis revealed an early activation of PSCs that preceded development of fibrosis 14. In mice, repeated intraperitoneal application of supraphysiological cerulein doses causes a pancreatic injury and, subsequently, fibrosis 1516. In agreement with the data mentioned above, collagen gene expression was colocalized to PSCs 16. Overexpression of transforming growth factor-beta (TGF-β) 1 in transgenic mice has been shown to be associated with increasing deposition of ECM in the pancreas. In parallel with the development of fibrosis, the number of PSCs in the pancreas increased 17.

Recently, it has also been suggested that PSCs contribute to regeneration early after acute necrotising pancreatitis in humans 18.

Together, in vitro and in vivo data suggest that PSCs are essentially involved in the development of pancreatic fibrosis.

Based on the results of various recent studies, extracellular factors involved in PSC activation may be divided into two major groups: (I) cytokines/growth factors 319202122 and (II) ethanol and its metabolites, most of all acetaldehyde 23.

Cytokines stimulating PSC activation include platelet-derived growth factor (PDGF) 3192122, the TGF-β family members TGF-β1 3192122 and activin A (24), TGF-alpha 322, basic fibroblast growth factor 322, tumor necrosis factor-α (TNF-α) 22, interleukin (IL)-1 20 and IL-6 20. While TGF-β1 efficiently promotes ECM synthesis 3192122, PDGF is considered to be the most effective mitogen 22. Furthermore, PDGF also enhances the migratory capacity of PSCs 25. Potential sources of cytokines stimulating PSC activation in the inflamed pancreas are, for example, activated macrophages (secreting TGF-β1) 26, platelets (containing PDGF and TGF-β1) 21, and possibly acinar cells (expressing, among other cytokines, TNF-α 27, IL-1 and IL-6 28). Importantly, PSCs themselves are capable of synthesizing cytokines such as TGF-β1 2930, activin A 24 and IL-1 31. These observations suggest the existence of autocrine loops that may contribute to the perpetuation of PSC activation after an initial exogenous signal, thereby promoting the development of fibrosis.

Recent studies have also implicated the pancreatic renin-angiotensin system 3233 in pancreatic fibrogenesis. Thus, application of the angiotensin-converting enzyme inhibitor lisinopril 34, as well as the angiotensin II receptor antagonist candesartan 35, suppressed pancreatic inflammation and fibrosis in an animal model of spontaneously occurring chronic pancreatitis, Wistar Bonn/Kobori rats. In angiotensin II receptor type 1a-deficient (AT1a(-/-)) mice, pancreatic fibrosis induced by repeated episodes of acute pancreatitis (following cerulein injections) was found to be attenuated 36. In vitro, angiotensin II (ATII) stimulates PSC proliferation 3738 and induces cell contraction 38.

Cytokines that act as antagonists of PSC activation have not been systematically studied so far. Recently, it has been shown that IFN-α protects hepatic stellate cells from lipid peroxidation by enhancing biological activities against oxidative stress, resulting in an inhibition of activation 39. Furthermore, antiproliferative effects of IFN-α 40, IFN-β and IFN-γ 41 on HSCs have been reported. On the other hand, IFN-α also inhibits spontaneous apoptosis of activated HSCs 40. The effects of interferons on pancreatic fibrogenesis remain to be characterized.

Although it is known for a long time that chronic pancreatitis, associated with fibrosis, is a serious complication of alcohol abuse, the pathogenesis of alcoholic pancreatitis still remains to be fully elucidated [reviewed in 42]. In recent studies, the question has been addressed how long-term alcohol consumption is linked to PSC activation and fibrosis. It has been proposed that the profibrogenic effects of ethanol are in part mediated by PSC-activating proinflammatory cytokines released during episodes of alcoholic pancreatitis (associated with necroinflammation) 43. Furthermore, in vitro data suggest that ethanol directly acts on PSCs and induces activation 23: Cultured PSCs respond to ethanol application by increased α-SMA expression and collagen synthesis. Stimulatory effects of ethanol were detectable both in already activated and still quiescent PSCs. The cells express alcohol dehydrogenase, indicating that they are capable of ethanol oxidation and generation of its metabolite acetaldehyde. Very likely, induction of oxidant stress in PSCs contributes to the profibrogenic effects of ethanol 23. Although the exact chain of events linking ethanol abuse to pancreatic inflammation and PSC activation remains to be described, it is likely that both direct and indirect (cytokine-mediated) effects of ethanol on PSCs are involved in the development of pancreatic fibrosis.

In the past two years, analysis of signal transduction pathways regulating PSC function has become a focus of attention. As detailed below, identification of signaling molecules that play a crucial role in PSC activation is a promising approach for the development of therapeutic strategies to inhibit pancreatic fibrosis. It is therefore envisaged that the systematic elucidation of signaling pathways in PSCs will also be one of the most important issues for future research. So far, research regarding intracellular signaling in PSCs has focused on two main aspects: the role of enzymes of the mitogen-activated protein kinase (MAPK) family and the transcriptional control of PSC activation.

MAPKs are a family of serine/threonine specific protein kinases with a wide range of biological functions in the regulation of fundamental cellular processes, including gene expression, proliferation and cell survival/apoptotic cell death 444546. In mammalian cells, three major MAPK families (extracellular signal-regulated kinases [ERKs], c-Jun N-terminal kinase [JNK] and p38) have been identified 45, and all of them have recently been studied with respect to the regulation of PSC activation. The best-characterized ERKs, ERK 1 and 2, are activated through a well-established pathway (induced by many growth factors) that involves, among several other cytosolic proteins, the small G-protein Ras and the serine/threonine-specific protein kinase Raf-1 45. In the process of PSC activation induced by sustained culture, ERK 1/2 activation is an early event that precedes exhibition of a myofibroblastic phenotype 47. The strong PSC mitogen PDGF induces an activation of ERK 1/2, and inhibition of signaling through the Ras-Raf-ERK signaling cascade attenuates PSC proliferation 474849. It has also been shown that exposure of PSCs to ethanol and acetaldehyde is accompanied by a fast 50 and long-lasting 51 ERK 1/2 activation.

The other two major MAP kinase pathways, involving JNK and p38, are well-established mediators of signals induced by pro-inflammatory cytokines and cellular stressors (e.g., oxidant stress, UV irradiation) 52. In PSCs, both JNK and p38 are activated in response to ethanol/acetaldehyde exposure 5051. Inhibition of p38 enzymatic activity interferes with ethanol-induced myofibroblastic transdifferentiation of PSCs 51. The p38 signaling pathway has also been implicated in the mediation of the mitogenic PDGF effect and in the induction of PSC activation induced by sustained culture 53. Incubation of freshly isolated PSCs with the JNK inhibitor SP600125 attenuates proliferation of the cultured cells 54. MAP kinase pathways have also been shown to be involved in ATII signaling in PSCs 3738. Together, these data support the hypothesis that MAPKs are key mediators of activation signals in PSCs.

Two other intracellular signal transduction pathways that have recently been studied regarding their role in PSC activation are the phosphatidylinositol 3 (PI 3)-kinase and the Rho-Rho kinase (ROCK) pathway. The results suggest that PI 3-kinase activity is required for PDGF-stimulated PSC migration but not proliferation 4955. The Rho-ROCK pathways was shown to be involved in the activation process of PSCs in vitro by regulating the actin cytoskeleton 56.

Cytokine and growth factor receptors exert their effects on the expression of target genes through signaling cascades that regulate the activity of a characteristic set of transcription factors. Recently, the group of the author has analyzed the activation profiles of activator protein (AP)-1 5758, signal transducer and activator of transcription (STAT) 3 59 and nuclear factor (NF)-κB 6061 in the course of PSC activation induced by sustained culture. AP-1 and NF-κB displayed an earlier maximum of DNA binding activity than STAT3 62. Further experiments revealed that phenotypic transition of PSCs towards myofibroblasts was accompanied by characteristic changes of AP-1 complex composition (increase of the JunD content relative to the one of JunB) 62. DNA binding of AP-1 in PSCs is induced by PDGF, suggesting AP-1 activation as an important step in the process of PSC activation 47.

In the transduction of TGF-β receptor-derived signals into the nucleus, Smad transcription factors play a central role 6364. Studies by Ohnishi and co-workers revealed that TGF-β1 stimulated PSC activation (indicated by increased α-SMA expression) in a Smad2-dependent manner, while Smad3 was required for TGF-β1-induced growth inhibition 65. Interestingly, exogenous TGF-β1 increased TGF-β1 mRNA expression in PSCs through an ERK-dependent but Smad2/3-independent pathway. Together, these data suggest distinct roles of Smad2-, Smad3- and ERK-dependent pathways in TGF-β1 regulation of PSC functions. Based on recently published data on HSC biology 66, it can be hypothesized that Smad7, a negative regulator of TGF-β signaling, might act as a transcriptional inhibitor of PSC activation, but so far experimental evidence has not been presented.

Recent studies have implicated the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) in the inhibition of stellate cell activation in liver 676869 and pancreas 69: The PPARγ ligands 15-deoxy-Δ12,14-prostaglandin J₂ and troglitazone (an antidiabetic drug of the thiazolidinedione group) act as antagonists of PSC activation in vitro that decrease cell proliferation and expression of α-SMA 70. In Wistar Bonn/Kobori rats, troglitazone attenuates pancreatic inflammation and fibrosis 71. The antifibrotic effect of the drug, however, was found to be in part mediated via a PPARγ-independent mechanism 72. Thus, the precise role of PPARγ in pancreatic fibrogenesis remains to be elucidated in further studies.

While the role of activated PSCs in pancreatic fibrosis is well established, the physiological functions of their quiescent precursors are less well understood. Importantly, PSCs are not only a source of ECM but also of matrix-degrading enzymes of the MMP (matrix metalloproteinases) family and their inhibitors (tissue inhibitors of matrix metalloproteinases, TIMPs). Thus, PSCs have been shown to secrete MMP-2, MMP-9 and MMP-13 and to express TIMP-1 and TIMP-2 73. It appears therefore likely that PSCs participate in the regulation of matrix turnover in the healthy pancreas.

The embryonic origin of PSCs still remains to be determined. Very recently, Seaberg et al. 74 reported the clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages, including β-like cells and pancreatic stellate cells. With regard to PSC biology, one implication of this pioneer study is that PSCs share with exocrine and endocrine pancreatic lineages a common progenitor cell. Kruse et al. 75 have described the isolation and culture of undifferentiated pancreatic cells, capable of extended self-renewal and spontaneous differentiation into cells of all three germ layers. The relationships between these cells, which were described as stellate-like cells, and PSCs are currently unknown and should be further studied using clonal cell populations.

Until now, the physiological consequences of vitamin A-storage in PSCs remain unclear. It has recently been shown by the group of the author that the vitamin A derivate all-trans retinoic acid has complex effects on PSC function and acts, at least in part, as an antagonist of the activation process 76. It is, therefore, tempting to speculate that retinoic acids, through the binding to their nuclear receptors and the regulation of gene expression, are involved in the maintenance of a quiescent PSC phenotype. In this scenario, the loss of retinoids in the course of PSC activation might be not an epiphenomenon but an essential prerequisite.

In the past, research regarding PSC biology has almost exclusively focused on the molecular basics of the activation process. However, given that participation in regeneration after pancreatic injury is an important function of activated PSCs, it is apparent that a disturbance of the inactivation or elimination of activated PSCs, rather than PSC activation itself, is the pathological process that leads to fibrosis. So far, it has not been systematically studied whether activated PSCs are capable of returning into a quiescent stage after fulfilling a repair function. Alternatively, elimination by apoptosis might be important in terminating the wound-healing response after pancreatic injury 77.

Finally, work on the complex relationships between PSCs and pancreatic tumor cells is still in its infancy. Very likely, activation of PSCs not simply accompanies tumor progression but plays an active role in this process. Thus, it has been recently been shown that pancreatic cancer growth and progression is accelerated through complex functional interactions between carcinoma cells and PSCs 78. Furthermore, the increased deposition of connective tissue in pancreatic carcinoma was suggested to be the result of a paracrine stimulation of PSCs by cancer cells 79. Interestingly, TGF-β1-transfected pancreatic tumor cells have been demonstrated to induce a rich stroma after orthotopical transplantation in the nude mouse pancreas 80. Considering the established role of TGF-β1 in PSC activation 3192122, it appears likely that the cytokine is a key effector in tumor-associated pancreatic fibrosis. It is easy to predict that the further analysis of PSC activation in pancreatic cancer will be an important research area in the future.

Studies on PSC biology are still hampered by the limited availibility of primary cells. Possibly, recently established pancreatic stellate cell lines 8182 will be helpful in overcoming this problem.

Given that activated PSCs are the principle effector cells in pancreatic fibrosis, targeting PSCs might become a promising therapeutic approach. Principle strategies that can be envisaged include an interruption/reversion of the activation process as well as an elimination of activated PSCs, e.g. through an induction of apoptosis. So far, potential antifibrotic drugs have been mainly tested in models of liver fibrosis (reviewed in 83). The existence of common mechanisms in the development of liver and pancreatic fibrosis (particularly, the key role of activated stellate cells), however, suggests that at least some of these drugs may also be effective inhibitors of fibrogenesis in the pancreas. In this regard, the efficiency of substances interfering with the action of stellate cell mitogens (e.g., PDFG), or cytokines stimulating ECM synthesis (especially TGF-β), should be tested in animal models of pancreatic fibrosis. The inhibitory effects of an angiotensin-converting enzyme inhibitor 34, as well as an ATII receptor antagonists 35, on pancreatic fibrosis need to be further evaluated. Interesting candidates are also cytokines that display inhibitory effects on hepatic stellate cell activation, such as interferons.

As described above, studies on the regulation of PSC activation at the intracellular level have identified key mediators of stimulatory and inhibitory signals. Targeting molecules such as PPARγ, MAP kinases, PI 3-kinase, or Smad proteins might become an important approach for the treatment of pancreatic fibrosis in the future. Further progress in the development of antifibrotic therapies can be expected from the ongoing elucidation of the molecular principles of PSC activation.