No abstract

The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.

SUMMARY: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 l/ml aPL increased collagen type I concentration in PSC supernatants by 1.99 Ϯ 0.17 times and c-FN of 2.49 Ϯ 0.28 times, mean Ϯ SD, n ϭ 3). nPL and aPL also significantly stimulated cell proliferation (increased bromodeoxyuridine (BrdU) incorporation by 6.4 Ϯ 0.78 times and 10 Ϯ 0.29 times, respectively). By preincubating aPL with transforming growth factor ␤ (TGF␤)-and platelet-derived growth factor (PDGF)-neutralizing antibodies and the TGF␤-latency associated peptide, respectively, TGF␤1 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 (TGF␤1) of cultured PSC. We suggest that platelets and PSC cooperate in the development of pancreas fibrosis. (Lab Invest 2000, 80:47-55).

Human cells acquire vitamin C using two different transporter systems, the sodium-ascorbic acid co-transporters with specificity for ascorbic acid, and the facilitative glucose transporters with specificity for dehydroascorbic acid. There is no information on the mechanism of vitamin C transport across the intestinal barrier, a step that determines the bioavailability of vitamin C in humans. We used the colon carcinoma cell line CaCo-2 as an in vitro model for vitamin C transport in enterocyte-like cells. The results of transport kinetics, sodium dependence, inhibition studies, and reverse transcriptase-PCR analysis indicated that CaCo-2 cells express the sodium-ascorbate co-transporters SVCT1 and SVCT2, the dehydroascorbic acid transporters GLUT1 and GLUT3, and a third dehydroascorbic acid transporter with properties expected for GLUT2. Analysis by real time quantitative PCR revealed that the post-confluent differentiation of CaCo-2 cells was accompanied by a marked increase (4-fold) in the steadystate level of SVCT1 mRNA, without changes in SVCT2 mRNA levels. Functional studies revealed that the differentiated cells expressed only one functional ascorbic acid transporter having properties expected for SVCT1, and transported ascorbic acid with a V max that was increased at least 2-fold compared with pre-confluent cells. Moreover, post-confluent Caco-2 cells growing as monolayers in permeable filter inserts showed selective sorting of SVCT1 to the apical membrane compartment, without functional evidence for the expression of SVCT2. The identification of SVCT1 as the transporter that allows vectorial uptake of ascorbic acid in differentiated CaCo-2 cells has a direct impact on our understanding of the mechanism for vitamin C transport across the intestinal barrier.

We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroen-terologyChronic pancreatitis is characterized by the presence of chronic inflammatory lesions, the destruction of exocrine parenchyma, and fibrosis. 1 Until now the pathobiochemical and molecular mechanisms resulting in pancreas fibrosis have been controversial and largely unknown. Fibroblast and fibroblast-like cell activation was reported to be a common observation in acute and chronic pancreatitis. [2][3][4][5][6] It has been suggested that periacinar fibroblast-like cells might play a role in fibrogenesis by synthesizing significant amounts of extracellular matrix (ECM), in particular collagens. 7,8 Studies from our group have shown that in the cerulein-induced pancreatitis model, proliferation of acinar and centroacinar cells is associated with an increase in mitotic activity of fibroblasts, which is followed by a stimulated synthesis and deposition of collagen. 5,9 In an earlier study, we illustrated an enhanced expression of transforming growth factor ␤ (TGF␤) during regeneration from cerulein-induced pancreatitis in acinar cells and stromal cells of the rat pancreas, which was followed by stimulated matrix synthesis. 10 Furthermore, we identified and characterized the matrix-producing cell type responsible for pancreas fibrosis, the pancreatic stellate cell (PSC) in mice, rats and humans. 11,12 Phenotypic transformation of PSCs is characterized by a disappearance of fat droplets and retinyl-esters, development of a prominent endoplasmic reticulum, enhanced expression of smooth muscle ␣-actin, and increased synthesis of collagen types I and III and fibronectin. 11 Pancreas fibrosis is commonly associated with chronic inflammation. 4 In chronic pancreatitis and in pancreas carcinoma, infiltrating mononuclear cells 13 might potentiate fibrogenesis by the release of cytokines stimulating stromal cells 14 and by their cytotoxic effects. 15 The decreased intestinal motility during pancreatitis causes a bacterial translocation to the peritoneal fluid, lymph, blood, liver, and pancreas. 16 Bacterial lipopolysaccharides (LPS) and the LPS-binding protein form a complex that binds to the surface receptor CD14 of the invaded monocytes, triggering the cells to become activated. 17,18 Supported by Bausteinfö rderung University of Ulm (P.347) and Deutsche Forschungsgemeinschaft (SFB 518, Project A7) to M. G. B.