Alain Vonlaufen

Pancreatic stellate cells: partners in crime with pancreatic cancer cells.

Pancreatic stellate cells (PSC) produce the stromal reaction in pancreatic cancer, but their role in cancer progression is not fully elucidated. We examined the influence of PSCs on pancreatic cancer growth using (a) an orthotopic model of pancreatic cancer and (b) cultured human PSCs (hPSC) and human pancreatic cancer cell lines MiaPaCa-2 and Panc-1. Athymic mice received an intrapancreatic injection of saline, hPSCs, MiaPaCa-2 cells, or hPSCs + MiaPaCa-2. After 7 weeks, tumor size, metastases, and tumor histology were assessed. In vitro studies assessed the effect of cancer cell secretions on PSC migration and the effect of hPSC secretions on cancer cell proliferation, apoptosis, and migration. Possible mediators of the effects of hPSC secretions on cancer cell proliferation were examined using neutralizing antibodies. Compared with mice receiving MiaPaCa-2 cells alone, mice injected with hPSCs + MiaPaCa-2 exhibited (a) increased tumor size and regional and distant metastasis, (b) fibrotic bands (desmoplasia) containing activated PSCs within tumors, and (c) increased tumor cell numbers. In vitro studies showed that, in the presence of pancreatic cancer cells, PSC migration was significantly increased. Furthermore, hPSC secretions induced the proliferation and migration, but inhibited the apoptosis, of MiaPaCa-2 and Panc-1 cells. The proliferative effect of hPSC secretions on pancreatic cancer cells was inhibited in the presence of neutralizing antibody to platelet-derived growth factor. Our studies indicate a significant interaction between pancreatic cancer cells and stromal cells (PSCs) and imply that pancreatic cancer cells recruit stromal cells to establish an environment that promotes cancer progression.

Role of Pancreatic Stellate Cells in Pancreatic Cancer Metastasis

Pancreatic stellate cells (PSCs) produce the stromal reaction in pancreatic cancer (PC), and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using both in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approaches. A sex mismatch study (injection of male hPSCs plus female PC cells into the pancreas of female mice) was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the Y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) quantifying human microvascular endothelial cell (HMEC-1) tube formation in vitro on exposure to conditioned media from hPSCs. Transendothelial migration was assessed in vitro by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs plus female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; and iii) exhibited transendothelial migration that was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis, and are able to intravasate/extravasate to and from blood vessels.

Pancreatic Stellate Cells and Pancreatic Cancer Cells: An Unholy Alliance

Pancreatic cancer--a tumor displaying a particularly abundant stromal reaction--is notorious for its poor prognosis. Recent studies, via newly developed orthotopic models, provide compelling evidence of an important role for pancreatic stellate cells (PSC) in pancreatic cancer progression. Characterization of the mechanisms mediating PSC-cancer interactions will lead to the development of much needed alternative therapeutic approaches to improve disease outcome.

The role of inflammatory and parenchymal cells in acute pancreatitis

The infiltration of inflammatory cells into the pancreas is an early and central event in acute pancreatitis that promotes local injury and systemic complications of the disease. Recent research has yielded the important finding that resident cells of the pancreas (particularly acinar and pancreatic stellate cells) play a dynamic role in leukocyte attraction via secretion of chemokines and cytokines and expression of adhesion molecules. Significant progress has been made in recent years in our understanding of the role of leukocyte movement (adhesion to the blood vessel wall, transmigration through the blood vessel wall and infiltration into the parenchyma) in the pathophysiology of acute pancreatitis. This review discusses recent studies and describes the current state of knowledge in the field. It is clear that detailed elucidation of the numerous processes in the inflammatory cascade is an essential step towards the development of improved therapeutic strategies in acute pancreatitis. Studies to date suggest that combination therapy targeting different steps of the inflammatory cascade may be the treatment of choice for this disease. Copyright

Molecular mechanisms of pancreatitis: current opinion.

Pancreatitis (necroinflammation of the pancreas) has both acute and chronic manifestations. Gallstones are the major cause of acute pancreatitis, whereas alcohol is associated with acute as well as chronic forms of the disease. Cases of true idiopathic pancreatitis are steadily diminishing as more genetic causes of the disease are discovered. The pathogenesis of acute pancreatitis has been extensively investigated over the past four decades; the general current consensus is that the injury is initiated within pancreatic acinar cells subsequent to premature intracellular activation of digestive enzymes. Repeated attacks of acute pancreatitis have the potential to evolve into chronic disease characterized by fibrosis and loss of pancreatic function. Our knowledge of the process of scarring has advanced considerably with the isolation and study of pancreatic stellate cells, now established as the key cells in pancreatic fibrogenesis. The present review summarizes recent developments in the field particularly with respect to the progress made in unraveling the molecular mechanisms of acute and chronic pancreatic injury secondary to gallstones, alcohol and genetic factors. It is anticipated that continued research in the area will lead to the identification and characterization of molecular pathways that may be therapeutically targeted to prevent/inhibit the initiation and progression of the disease.

Expression of the bile acid receptor FXR in Barrett's esophagus and enhancement of apoptosis by guggulsterone in vitro

BackgroundBarretts esophagus, a risk factor for esophageal adenocarcinoma, is associated with reflux disease. The aim of this study was to assess the expression of bile acid receptors in the esophagus (normal, esophagitis, Barretts esophagus and adenocarcinoma) and to investigate their possible function.Resultsthe expression of the bile acid receptors FXR and VDR in esophageal biopsies from patients with a normal mucosa, esophagitis, Barretts esophagus or adenocarcinoma (n = 6 per group) and in cell lines derived from Barretts esophagus and esophageal adenocarcinoma, was assessed by real time Q-PCR and immunohistochemistry. The effect of guggulsterone, an antagonist of bile acid receptors, on apoptosis of Barretts esophagus-derived cells was assessed morphologically, by flow cytometry and by measuring caspase 3 activity.The expression of FXR was increased in esophagitis, Barretts esophagus and adenocarcinoma compared to normal mucosa by a mean of 44, 84 and 16, respectively. Immunohistochemistry showed a weak expression in normal esophagus, a strong focal reactivity in Barretts esophagus, and was negative in adenocarcinoma. VDR expression did not significantly differ between groups. In cell cultures, the expression of FXR was high in Barretts esophagus-derived cells and almost undetectable in adenocarcinoma-derived cells, whereas VDR expression in these cell lines was not significantly different. In vitro treatment with guggulsterone was associated with a significant increase in the percentage of apoptotic cells and of the caspase 3 activity.Conclusionthe bile acid receptor FXR is significantly overexpressed in Barretts esophagus compared to normal mucosa, esophagitis and esophageal adenocarcinoma. The induction of apoptosis by guggulsterone in a Barretts esophagus-derived cell line suggests that FXR may contribute to the regulation of apoptosis.

Pancreatic stellate cells produce acetylcholine and may play a role in pancreatic exocrine secretion

The pancreatic secretagogue cholecystokinin (CCK) is widely thought to stimulate enzyme secretion by acinar cells indirectly via activation of the vagus nerve. We postulate an alternative pathway for CCK-induced pancreatic secretion. We hypothesize that neurally related pancreatic stellate cells (PSCs; located in close proximity to the basolateral aspect of acinar cells) play a regulatory role in pancreatic secretion by serving as an intermediate target for CCK and secreting the neurotransmitter acetylcholine (ACh), which, in turn, stimulates acinar enzyme secretion. To determine whether PSCs (i) exhibit CCK-dependent ACh secretion and (ii) influence acinar enzyme secretion, primary cultures of human and rat PSCs were used. Immunoblotting and/or immunofluorescence was used to detect choline acetyltransferase (ACh synthesizing enzyme), vesicular ACh transporter (VAChT), synaptophysin, and CCK receptors 1 and 2. Synaptic-like vesicles in PSCs were identified by EM. ACh secretion by PSCs exposed to 20 pM CCK was measured by LC-MS/MS. Amylase secretion by acini [pretreated with and without the muscarinic receptor antagonist atropine (10 μM) and cocultured with PSCs] was measured by colorimetry. PSCs express ACh synthesizing enzyme, VAChT, synaptophysin, and CCK receptors; exhibit CCK-dependent ACh secretion; and stimulate amylase secretion by acini, which is blocked by atropine. In conclusion, PSCs express the essential elements for ACh synthesis and secretion. CCK stimulates ACh secretion by PSCs, which, in turn, induces amylase secretion by acini. Therefore, PSCs may represent a previously unrecognized intrapancreatic pathway regulating CCK-induced pancreatic exocrine secretion.

Function of both sinusoidal and canalicular transporters controls the concentration of organic anions within hepatocytes

We hypothesized that the function of both sinusoidal and canalicular transporters importantly controls the concentrations of organic anions within normal hepatocytes. Consequently, we investigated how acute transport regulation of the sinusoidal organic anion transporting polypeptides (Oatps) and the canalicular multidrug resistance associated protein 2 (Mrp2) determines the hepatic concentrations of the organic anion gadolinium benzyloxypropionictetraacetate (BOPTA) in rat livers. Livers were perfused with labeled BOPTA in different experimental settings that modify the function of Oatps and Mrp2 through the protein kinase C (PKC) pathway. Intrahepatic concentrations were continuously measured with a gamma probe placed above rat livers. Labeled BOPTA was also measured in perfusate and bile. We showed that when the function of Oatps and Mrp2 is modified in such a way that BOPTA entry and exit are similarly decreased, concentrations of organic anions within hepatocytes remain unaltered. When exit through Mrp2 is abolished, hepatic concentrations are high if entry through Oatps is only slightly decreased (livers without Mrp2 expression) or low if BOPTA uptake is more importantly decreased (livers perfused with a PKC activator). These results highlight that the function of both sinusoidal and canalicular transporters is important to determine the concentration of organic anions within hepatocytes.

Withdrawal of alcohol promotes regression while continued alcohol intake promotes persistence of LPS-induced pancreatic injury in alcohol-fed rats

Background and aims Administration of repeated lipopolysaccharide (LPS) injections in alcohol-fed rats leads to significant pancreatic injury including fibrosis. However, it remains unknown whether alcoholic (chronic) pancreatitis has the potential to regress when alcohol is withdrawn. The aims of the study were (1) to compare the effect of alcohol withdrawal/continuation on pancreatic acute injury and fibrosis; and (2) to assess the effects of alcohol ± LPS on pancreatic stellate cell (PSC) apoptosis in vivo and in vitro. Methods Rats fed isocaloric Lieber–DeCarli liquid diets ± alcohol for 10 weeks were challenged with LPS (3 mg/kg/week for 3 weeks) and then either switched to control diet or maintained on an alcohol diet for 3 days, 7 days or 3 weeks. Pancreatic sections were assessed for acute tissue injury, fibrosis, PSC apoptosis and activation. Cultured rat PSCs were exposed to 10 mM ethanol ± 1 μg/ml LPS for 48 or 72 h and apoptosis was assessed (Annexin V, caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)). Results Withdrawal of alcohol led to resolution of pancreatic lesions including fibrosis and to increased PSC apoptosis. Continued alcohol administration perpetuated pancreatic injury and prevented PSC apoptosis. Alcohol and LPS significantly inhibited PSC apoptosis in vitro, and the effect of LPS on PSC apoptosis could be blocked by Toll-like receptor 4 small interfering RNA. Conclusions Induction of PSC apoptosis upon alcohol withdrawal is a key mechanism mediating the resolution of pancreatic fibrosis. Conversely, continued alcohol intake perpetuates pancreatic injury by inhibiting apoptosis and promoting activation of PSCs. Characterisation of the pathways mediating PSC apoptosis has the potential to yield novel therapeutic strategies for chronic pancreatitis.

The role of junctional adhesion molecule C (JAM-C) in acute pancreatitis.

The recruitment of inflammatory cells contributes significantly to tissue injury in acute pancreatitis. This process implies several molecular interactions between circulating and endothelial cells. The adhesion molecule junctional adhesion molecule C (JAM‐C) is involved in leukocyte transendothelial migration and it can form homophilic (JAM‐C/JAM‐C) and heterophilic interactions with the leukocyte integrin αMβ2. In this study, the effect of early administration of monoclonal antibodies directed against JAM‐C in cerulein‐induced acute pancreatitis was assessed. This reagent significantly blocked influx of leukocytes, release of serum amylase, secretion of inflammatory cytokines, and acinar cell necrosis. These effects were rapid and protected against tissue injury throughout the duration of the model. Conversely, cerulein‐induced acute pancreatitis was more severe in transgenic mice overexpressing JAM‐C on endothelial cells under the control of the Tie2 promoter. It is proposed that JAM‐C expressed by endothelial cells contributes to the pathophysiology of acute pancreatitis and could be considered a target for clinical applications. Copyright