Marco Siech

Identification, culture, and characterization of pancreatic stellate cells in rats and humans☆☆☆

BACKGROUND & AIMS Until now, the basic matrix-producing cell type responsible for pancreas fibrosis has not been identified. In this report, retinoid-containing pancreatic stellate cells (PSCs) in rat and human pancreas are described, and morphological and biochemical similarities to hepatic stellate cells are shown. METHODS Electron and immunofluorescence microscopy (collagen types I and III, fibronectin, laminin, alpha-actin, and desmin) was performed using pancreatic tissue and cultured PSCs. Extracellular matrix synthesis was shown using quantitative immunoassay and Northern blot analysis. RESULTS PSCs are located in interlobular areas and in interacinar regions. Early primary cultured PSCs contain retinol and fatty acid retinyl-esters. Addition of retinol to passaged cells resulted in retinol uptake and esterification. During primary culture, the cells changed from a quiescent fat-storing phenotype to a highly synthetic myofibroblast-like cell expressing iso-alpha-smooth muscle actin (>90%) and desmin (20%-40%) and showing strong positive staining with antibodies to collagen types I and III, fibronectin, and laminin. As determined on protein and messenger RNA level, serum growth factors stimulated the synthesis of collagen type I and fibronectin. CONCLUSIONS The identification of PSCs, particularly in fibrotic areas, and the similarities of these cells to hepatic stellate cells suggest that PSCs participate in the development of pancreas fibrosis.

Platelet-Derived Growth Factors Stimulate Proliferation and Extracellular Matrix Synthesis of Pancreatic Stellate Cells: Implications in Pathogenesis of Pancreas Fibrosis

At present, the cell-cell interactions and molecular mechanisms of pancreas fibrogenesis are largely unknown. The purpose of this study was to investigate paracrine stimulatory loops between platelets and pancreatic stellate cells (PSC). Human PSC were obtained by outgrowth from fibrotic human pancreas. Native platelet lysate (nPL) and transiently acidified platelet lysate (aPL) were added to cultured PSC (passage 4 to 7) in the absence of serum. The synthesis of collagen types I and III and c-fibronectin (cFN) was demonstrated on protein (immunofluorescence and quantitative immunoassay) and mRNA (Northern blot) level. Using sections of human pancreas with acute pancreatitis, platelet aggregates in capillaries were demonstrated by transmission electron microscopy. nPL, and to an even greater extent aPL, significantly increased the synthesis of collagen types I and III and of c-FN (120 μl/ml aPL increased collagen type I concentration in PSC supernatants by 1.99 ± 0.17 times and c-FN of 2.49 ± 0.28 times, mean ± sd, n = 3). nPL and aPL also significantly stimulated cell proliferation (increased bromodeoxyuridine (BrdU) incorporation by 6.4 ± 0.78 times and 10 ± 0.29 times, respectively). By preincubating aPL with transforming growth factor β (TGFβ)- and platelet-derived growth factor (PDGF)-neutralizing antibodies and the TGFβ-latency associated peptide, respectively, TGFβ1 was identified as the main mediator stimulating matrix synthesis and PDGF as the responsible mitogen. Our data demonstrate that platelets contain fibrogenic mediators that stimulate proliferation (PDGF) and matrix synthesis (TGFβ1) of cultured PSC. We suggest that platelets and PSC cooperate in the development of pancreas fibrosis.

CYSTIC TUMOURS OF THE PANCREAS : DIAGNOSTIC ACCURACY, PATHOLOGIC OBSERVATIONS AND SURGICAL CONSEQUENCES

Background: Cystic neoplasms of the pancreas account for only 1% of primary pancreatic lesions. However, patients with these tumors are diagnosed more frequently. Up to now, nonsurgical management is still the established form of treatment of benign cystic tumours of the pancreas. Methods: Between 1987 and 1996 we treated 51 patients with serous and mucinous cystadenoma and their malignant counterparts, serous and mucinous cystadenocarcinoma. Results: Eighty-five percent of the patients presented symptoms. Computed tomography and endoscopic cholangiopancreatography (ERCP) were the most sensitive diagnostic techniques; however, in three patients with serous cystadenoma and in one patient with serous cystadenocarcinoma, ERCP findings were completely normal. The tumour was resected in all but one patient. There was no perioperative mortality. After dismissal from the hospital, all patients in whom benign tumours had been resected are still alive; however, the late mortality of mucinous cystadenocarcinoma was 36% after a median follow-up of 6 years. Conclusion: Surgical resection is recommended in all cystic tumours, even in serous cystic tumours, because symptoms may develop and malignant transformation to serous cystadenocarcinoma is possible.

Pancreatic stellate cells are an important source of MMP-2 in human pancreatic cancer and accelerate tumor progression in a murine xenograft model and CAM assay

The effect of the characteristic desmoplastic reaction of pancreatic cancer on tumor progression is largely unknown. We investigated whether pancreatic stellate cells, which are responsible for the desmoplastic reaction, support tumor progression. Immunohistology revealed that matrix metalloproteinase-2 (MMP-2), which is suggested to promote pancreatic cancer progression, is present in stellate cells adjacent to cancer cells. In vitro, stellate cells exhibited a much higher basal expression of MMP-2 compared with cancer cells. Panc1-, MiaPaCa2- and SW850-conditioned media stimulated MMP-2 release of stellate cells as detected by zymography. Cancer cells expressed and released basigin [BSG, extracellular matrix metalloproteinase inducer (EMMPRIN), CD147], a glycoprotein that is known to stimulate MMP-2 in mesenchymal cells, as detected by immunostaining, western blot and reverse transcription-polymerase chain reaction. Tumor cell-conditioned medium and BSG purified by affinity chromatography from supernatants of cancer cells, but not supernatants depleted from BSG, stimulated expression of MMP-1 and MMP-2 of stellate cells as demonstrated by western blot and zymography. Moreover, the interaction of stellate cells and cancer cells promoted the invasiveness of Panc-1 cells in the chorioallantoic membrane assay and increased the weight of tumors induced by all carcinoma cell lines in nude mice by 2.1-3.7-fold. Our findings support the assumption that the interaction of stellate cells and cancer cells promotes progression of pancreatic cancer.

Transcriptome analysis of human hepatic and pancreatic stellate cells: organ-specific variations of a common transcriptional phenotype.

Pancreatic stellate cells (PSCs) are thought to be the primary source of the extensive fibrotic reaction characteristic of pancreatic cancer and chronic pancreatitis in humans. PSCs share many morphological and functional characteristics with hepatic stellate cells (HSCs), whose central role in liver fibrosis is well established. However, it has remained unclear if hepatic and pancreatic stellate cells are derived from a common cell lineage and if they are completely similar or if they possess organ-specific features. We have analysed the transcriptomes of HSCs, PSCs and skin fibroblasts to assess how the transcriptional phenotype of stellate cells differs from that of a typical fibroblast lineage cell and if there is evidence for a common stellate cell precursor. To this end, we have performed expression profiling of primary cultures of human HSCs, PSCs and skin fibroblasts using 23,000-feature ‘whole genome’ oligonucleotide micro-arrays. Expression data were verified using real-time PCR. The expression profiles of HSCs and PSCs displayed a great extent of similarity, clearly separating them from the fibroblasts. Predominantly extracellular and cell surface genes, but also signalling molecules, transcription factors and novel neural markers, were concordantly expressed in both stellate cell types. Despite this high degree of similarity, distinct differences in expression patterns were observed between HSCs and PSCs, reflecting organ-specific variations of the common stellate cell-specific phenotype.

Embolization for gastrointestinal hemorrhages

Abstract. Retrospective evaluation of interventional embolization therapy in the treatment of gastrointestinal hemorrhage over a long-term observation period from 1989 to 1997. Included in the study were 35 patients (age range 18–89 years) with gastrointestinal bleeding (GI) referred for radiological intervention either primarily or following unsuccessful endoscopy or surgery. Sources of GI bleeding included gastric and duodenal ulcers (n = 7), diverticula (n = 3), erosion of the intestinal wall secondary to malignancy (n = 6), vascular malformations (n = 4), and hemorrhoids (n = 2), as well as from postoperative (n = 6), posttraumatic (n = 2), postinflammatory (n = 4) or unknown (n = 1) causes. Ethibloc (12 cases) or metal coils (14 cases) were predominantly used as embolisates. In addition, combinations of tissue adhesive and gelfoam particles and of coils and Ethibloc were used (six cases). Finally, polyvinyl alcohol particles, a coated stent, and an arterial wire dissection were utilized in one case each. Bleeding was stopped completely in 29 of 35 cases (83 %). In one case (3 %) the source of bleeding was recognized but the corresponding vessel could not be catheterized. In five other cases (14 %) there was partial success with reduced, though still persistent, bleeding. The rate of complications was 14 %, including four instances of intestinal ischemia with fatal outcome in the first years, and, later, one partial infarction of the spleen without serious consequences. Gastrointestinal hemorrhage can be controlled in a high percentage of patients, including the seriously ill and those who had previously undergone surgery, with the use of minimally invasive interventional techniques. The availability of minicoils instead of fluid embolization agents has reduced the risk of serious complications.

Lipopolysaccharide-Activated Macrophages Stimulate the Synthesis of Collagen Type I and C-Fibronectin in Cultured Pancreatic Stellate Cells

We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421-435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 +/- 0.09-fold of control and c-fibronectin 1.89 +/- 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 +/- 0.2-fold and 2.80 +/- 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(alpha-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFbeta1/microgram of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFbeta1/microgram of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFbeta as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFbeta1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis.

Serous cystadenocarcinoma of the pancreas

SummaryThe mucinous type of cystadenoma of the pancreas is known to have malignant potential, whereas the serous type is believed to be benign. Therefore, the therapeutic strategies for serous cystadenomas are less aggressive than in mucinous cystadenoma, where complete operative resection is the procedure of choice.A patient with a biopsy-proven serous cystadenocarcinoma with a lymph node metastasis is reported. Considering the reported case and a review of the literature, the origin of serous cystadenocarcinoma appears to be from cystadenoma lesions.Even if this development of cystadenocarcinoma in pre-existing serous cystadenoma lesions is a rare entity, a less aggressive treatment of this lesion should be avoided in favor of resection procedures because of the good chance of cure.

Intraductal papillary mucinous tumor of the pancreas

BACKGROUND Since 1996 the classification of pancreatic tumors was replaced by the new World Health Organization nomenclature. Formerly mucinous cystadenomas are now distinguished between intraductal papillary mucinous tumors of the pancreas (IPMT) and mucinous cystadenomas. METHODS We reevaluated the pathological specimen and surgical therapy of 23 consecutive patients and followed up these patients up for 4 years in median. Between 1987 and 1997 we treated 8 patients with IPMT and 15 patients with mucinous cystadenomas. RESULTS Eighty-five per cent of all patients were symptomatic. Ultrasonography and computed tomography were the most sensitive diagnostic techniques. In 25%, the entire pancreas was involved with IPMT; that was not the case in any of the patients with mucinous cystadenoma. All patients were resected with no perioperative mortality. After dismissal from the hospital, all resected patients are still alive after a median follow-up of 4 years. In no patient with IPMT, but in 1 patient with mucinous cystadenoma, the tumor recurred. CONCLUSION Surgical resection is the treatment of choice in all cystic tumors, and the late outcome of IPMT is as good as for mucinous cystadenoma.

Pancreatic stellate cells--role in pancreas cancer.

BackgroundAdenocarcinomas of the pancreas are characterized by a rapid progression, an early metastasis, a limited response to chemo- and radiotherapy, and an intense fibrotic reaction known as tumor desmoplasia. Carcinoma cells are surrounded by a dense stroma consisting of myofibroblast-like cells, collagens, and fibronectin.Materials and methodsThis review describes the interaction of activated pancreatic stellate cells (myofibroblast-like cells) with tumor cells in pancreas adenocarcinomas. Our data were obtained in cell culture experiments and in in vivo investigations.ResultsCarcinoma cells produce soluble mediators and stimulate motility, proliferation, matrix-, and MMP synthesis of stellate cells. Vice versa-activated stellate cells release mitogens, stimulating proliferation of cancer cells. Cancer cell proliferation and resistance to apoptosis might further be induced by the microenvironment (extracellular matrix), which is primarily provided by stellate cells. A very important aspect in the interaction of stellate cells with cancer cells is the expression of EMMPRIN (extracellular matrix metalloproteinase inducer) by cancer cells, the shedding of the extracellular part of EMMPRIN by matrix metalloproteinases (MMPs), and the induction of MMPs in stellate cells by soluble EMMPRIN. In particular, the stellate cells in close proximity to tumor cells therefore express MMPs and degrade connective tissue.ConclusionThrough complex interactions between stellate cells and carcinoma cells, tumor progression and cancer cell invasion are accelerated. As we gain better understanding of these mechanisms, adequate therapies to reduce tumor cell invasion and cancer progression might be developed.