Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

Eric Schneider1,2,*, Alexandra Schmid-Kotsas1,*, Jinshun Zhao1, Hans Weidenbach2, Roland M. Schmid2, Andre Menke2, Guido Adler2, Johannes Waltenberger3, Adolf Grünert1, and Max G. Bachem1

Departments of 1 Clinical Chemistry and Pathobiochemistry, 2 Internal Medicine I, and 3 Internal Medicine II, University of Ulm, 89070 Ulm, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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.

pancreas fibrosis; growth factors; fibrogenic mediators; collagen; fibronectin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

CHRONIC PANCREATITIS is defined by the presence of chronic inflammation, destruction of acinar and ductal cells, intra- and perilobular fibrosis, and finally the irreversible scarring of parenchyma (14). Until now the molecular mechanisms and cell-cell interactions resulting in pancreas fibrosis were largely unknown. However, the progressive appearance of fibrotic tissue occurs regardless of the initiating triggers (45), a result of an increased deposition and a reduced degradation of extracellular matrix (22, 31, 45, 46). In contrast to fibrogenesis in the pancreas, liver fibrogenesis has been studied extensively during the past two decades. It is now generally accepted that hepatic stellate cells (HSC), formerly named perisinusoidal fat-storing cells and Ito cells, play a central role in liver fibrogenesis. In experimental and human liver injury, HSC change their phenotype from a quiescent retinoid-storing cell to a highly active and "synthetic" myofibroblast-like cell producing the majority of extracellular matrix, including collagen types I, III, and IV, fibronectin, and proteoglycan matrix (19, 24). Years ago, the existence of retinoid-containing fat-storing cells was demonstrated in pancreas of mice (50), rats, and humans (28). Although a potential role of these cells in pancreas remodeling and fibrosis was already discussed by Ikejiri (28), it took another eight years to isolate and characterize these cells (1, 8). Recently obtained data demonstrate that the fat-storing cells of the pancreas, now named pancreatic stellate cells (PSC), share homologies to HSC (8), including storage of retinyl palmitate, retinol esterification, expression of the cytofilaments desmin and in particular SMA, and phenotypic transition to a highly active matrix producing myofibroblast-like cells (1, 2, 8, 26). We have shown that extracellular matrix synthesis of PSC is stimulated by soluble mediators released by activated macrophages (43) and aggregating platelets (34). Others have shown that acetaldehyde, which is produced primarily in the liver by ethanol oxidation, directly stimulates matrix synthesis of cultured PSC (3). The objective of the present study was to identify polypeptide fibrogenic mediators stimulating proliferation and/or matrix synthesis of PSC.

The pattern of the growth factors to be studied was selected according to 1) published matrix synthesis-stimulating effects of certain growth factors in other cell types (6, 9, 13, 21, 37, 41), 2) the histopathological appearance of the growth factor or its precursor in chronic pancreatitis (20, 32, 44), and 3) data obtained in transgenic mice that show the development of fibrosis in animals overexpressing transforming growth factor (TGF)-beta 1 and TGF-alpha in the pancreas (11, 33). Therefore, we studied the effects of the polypeptide growth factors basic fibroblast growth factor (bFGF), TGF-alpha , TGF-beta 1, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)-I, IGF-II, and tumor necrosis factor (TNF)-alpha , added alone and in combination, on cell activation (expression of iso-alpha -smooth actin), proliferation, and matrix synthesis of cultured rat PSC. We show that TGF-beta 1 and TNF-alpha stimulate the change in the cell phenotype. In addition, using the activated phenotype of cultured rat PSC (myofibroblast-like cells), we show that PDGF represents the most effective mitogen and bFGF and TGF-beta 1 represent fibrogenic mediators stimulating extracellular matrix synthesis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Reagents were purchased from the following sources: acetone from Merck (Darmstadt, Germany); bis-benzimide from Aventis (Frankfurt, Germany); FCS, amphotericin B, anti-smooth muscle alpha -actin (SMA), TGF-beta 1 (human recombinant), bFGF (human recombinant), PDGF-AB (human recombinant), TGF-alpha (human recombinant), High Pure RNA Extraction Kit, LightCycler FastStart SYBR Green and LightCycler DNA Master SYBR Green I from Roche (Mannheim, Germany); gelatin type B, ethidium bromide, bovine albumin fraction V, ethanol, (+)-L-ascorbic acid, calf thymus DNA, yeast tRNA, and RedTaq DNA Polymerase from Sigma (Deisenhofen, Germany); agarose, L-glutamine, and Superscript from GIBCO-BRL (Paisley, Scotland); TNF-alpha (human recombinant), IGF-I (human recombinant), and IGF-II (human recombinant) from Laboserv Diagnostic (Giessen, Germany); Delfia Eu-labeled streptavidin, Delfia Eu-labeled anti-rabbit IgG, and enhancement solution from PE-Life-Science-Wallac (Turku, Finland); rabbit anti-rat collagen type I, rabbit anti-laminin, and rabbit anti-rat collagen type III from Chemicon International (Temecula, CA); polyclonal rabbit anti-fibronectin from Dade-Behring (Marburg, Germany); poly(dA/dU) homopolymer from Pharmacia LKB (Freiburg, Germany); Hybond-N membrane, Hybond-C-extra membrane, and Random-Priming Kit (RPN 1601Y) from Amersham-Buchler (Braunschweig, Germany); anti-bromodeoxyuridine (BrdU), horseradish peroxidase (HRP)-anti-rabbit, FITC-conjugated streptavidin, biotinylated rabbit anti-mouse IgG, rabbit anti-vimentin, and HRP-conjugated streptavidin from Dako (Glostrup, Denmark); trypsin-EDTA, DMEM-Ham's F-12 medium, and penicillin from Biochrom (Berlin, Germany); Tyramide Signal Amplification (TSA)-Indirect from NEN Life Science Products (Boston, MA); GelStar from Biozym (Oldendorf, Germany); RNeasy Kit and QIAQuick Gel Extraction Kit from Qiagen (Hilden, Germany); cDNA probes for collagen-alpha 1 I, collagen-alpha 1 III, laminin, and fibronectin from American Type Culture Collection; oligonucleotide primers were synthesized by Interactiva (Ulm, Germany); and X-ray film was from Kodak (Rochester, NY). The ZnR5 cells heterotopically expressing the human PDGF beta -receptor were a generous gift from Carl-Henrik Heldin (Uppsala, Sweden).

Cell Isolation and Culture

Cells were isolated either by density gradient centrifugation after collagenase digestion of rat pancreas or by the outgrowth method using pancreas tissue of rats with cerulein pancreatitis. To obtain cells with the inactivated fat-storing phenotype, cells were isolated from the pancreas of untreated male Wistar rats (200-300 g). After the animals were anesthetized with pentobarbital sodium, the abdomen was opened, the common bile duct was ligated, and a cannula was inserted in the biliopancreatic duct. The rats were exsanguinated, and 1 ml collagenase P (6 mg/5 ml) containing McCoy's 5A medium (6 mg/5 ml) was instilled intraductally. The distended pancreas was removed, minced, and shaken in 4 ml collagenase P solution in an Erlenmeyer flask (37°C, 20 min). Thereafter, minced pancreas was incubated three times for 3 min with EDTA containing McCoy's 5A medium followed by three washing steps with McCoy's 5A medium. Thereafter, a second digestion was performed using 5 ml McCoy's 5A medium with 6 mg collagenase P, 5 mg hyaluronidase, 2 mg chymotrypsin, and 250 µl DNase (37°C, 20 min). Dispersion was accomplished by up-and-down suction through cannulas with decreasing diameters. After dissociation, the suspension was filtered through a 100-µm Nylon filter (Cell Strainer; Becton-Dickinson Labware, Franklin Lakes, NJ), and the filtrate was placed on top of 7.5 ml McCoy's 5A medium with 15 mg BSA and centrifuged for 5 min at 350 g. Thereafter, supernatant was removed, and the cell pellet was resuspended in Ham's F-12-DMEM (1:1, vol/vol) with 10% FCS. This cell suspension was layered on top of a Percoll-McCoy's 5A medium (7.5:2.5, vol/vol) density gradient and centrifuged for another 5 min at 180 g. Once centrifuged, cells were collected from the top of the gradient, washed, suspended in Ham's F-12-DMEM (1:1, vol/vol) with 20% FCS, antibiotics, amphotericin, and L-glutamine, counted, and seeded in a density of 0.5 × 104 cells/cm2.

The purity of the isolated cells (as assessed by light, phase-contrast, fluorescence, and electronmicroscopy) was ~80%. With the first (24 h after seeding) medium change, most of the contaminating cells were removed, and the cell cultures were almost (>95%) free of impurities. To demonstrate PSC activation (SMA, collagens I and III and fibronectin immunofluorescence, and real-time quantitative RT-PCR) and to study the expression of fibronectin splice variants, cells were cultured in the presence of 10% FCS beginning 24 h after seeding. To demonstrate the effect of growth factors on PSC activation, cells were cultured in the presence of 0.1% FCS.

To obtain higher numbers of cells, male Wistar rats weighing between 200 and 450 g were injected intraperitoneally with supramaximal doses (10 µg · kg-1 · h-1) of cerulein. The animals had free access to pelleted food and tap water. The rats were killed 2 days later by exsanguination under ether anesthesia. The pancreas was removed, placed directly in DMEM-Ham's F-12 supplemented with 10% FCS, 4 mmol/l L-glutamine, 100 U/ml penicillin, and 100 U/ml amphotericin, and then cut into small pieces of ~1 mm3 under sterile conditions. The pieces were placed in six-well plates and allowed to settle and adhere to the bottom of the plates in 4 ml DMEM-Ham's F-12 supplemented with 10% FCS. The plates were placed in a humidified atmosphere with 5% CO2 at 37°C. Medium was changed after 12 and 24 h. During the next 3-5 days, cells grew out from the tissue blocks and formed a confluent layer. After reaching confluence, monolayers were trypsinized and passaged 1:3. Cell populations between passages 2 and 6 were used to study the effects of growth factors on cell proliferation and matrix synthesis.

Determination of Cell Proliferation

PSC proliferation was quantified as described previously (34) by time-resolved fluorescence of a Europium-chelate after BrdU labeling. Furthermore, DNA was measured as previously described (8) by fluorometry using bis-benzimide and calf thymus DNA as a standard. DNA measurements (standards, controls, and samples) were done in triplicate.

Determination of Extracellular Matrix Synthesis

Fibronectin concentration. Fibronectin concentration in PSC supernatants was measured by time-resolved fluorescence immunoassay as described (8, 34, 43). All measurements (standards, controls, and samples) were done in duplicate. Variations of duplicate measurements were usually between 0.5 and 5% and did not exceed 8%. Fibronectin concentration was calculated on the basis of DNA.

Immunofluorescence microscopy of collagen types I and III, fibronectin, and SMA. To demonstrate cell-associated collagen types I and III and fibronectin immunofluorescence, microscopy was performed as described with minor modifications (8, 34, 43). For collagens, the staining sequence was rabbit-anti-rat-collagen I or III (1:50), biotin-anti-rabbit (1:100); streptavidin-HRP (1:100), biotin-TSA reagent (1:40), and streptavidin-FITC (1:100). The staining sequence for fibronectin was primary antibody (rabbit-anti-fibronectin, 1:50), secondary antibody (biotin-anti-rabbit, 1:100), and streptavidin-FITC (1:100). For SMA, the staining sequence was primary antibody (mouse anti-SMA, 1:50), secondary antibody (HRP-anti-mouse, 1:50), biotin-TSA reagent (1:40), and streptavidin-FITC (1:100). Each incubation step was followed by rinsing (3 × 5 min with PBS). Staining was observed using epifluorescence microscopy (C. Zeiss, Oberkochen, Germany), and photographs were taken using Ektachrome 400 film (Kodak). To compare different staining intensities, exposure time was always the same. Nonspecific staining was controlled by including rabbit or mouse nonimmune serum instead of specific first antibody.

Real-Time Quantitative RT-PCR and Conventional RT-PCR

Real-time quantitative RT-PCR was used to measure the steady-state levels of the mRNA of beta -actin, SMA, TGF-beta 1, connective tissue growth factor (CTGF), and fibronectin, and conventional RT-PCR was used to demonstrate fibronectin splice variants. Total RNA was extracted using a High Pure RNA Isolation Kit according to the manufacturer's instructions. The concentration and purity of RNA was determined by measuring the absorbance at 260 and 280 nm. For detection of beta -actin, SMA, TGF-beta 1, CTGF, and main fibronectin splice variant (EIII-A and EIII-B) mRNA, 570 ng of total RNA were reverse transcribed into cDNA with 0.02 U/ml RT at 42°C for 50 min in standard buffer according to the manufacturer's instructions. The following oligonucleotide primers were used: beta -actin sense primer 5'-GTGGGGCGCCCCAGGCACCA-3', antisense primer 5'-CTCCTTAATGTCACGCACGATTTC-3' (accession no. M10277); fibronectin sense primer 5'-TGTCACCCACCACCTTGA-3', antisense primer 5'-CTGATTGTTCTTCAGTGCGA-3' (accession no. L00191); TGF-beta sense primer 5'-AGTGGATCCACGAGCCCAA-3', antisense primer 5'-AGGAGCGCACGATCATGTT-3' (accession no. X02812); CTGF sense primer 5'-GCGTCTGCGCCAAGCA-3', antisense primer 5'-TGGACCAGGCAGTTGGCT-3' (accession no. M92934); SMA sense primer 5'-TGCTGGACTCTGGAGATG-3', antisense primer 5'-GTGATCACCTGCCCATC-3' (accession no. X06801); fibronectin EIII-A sense primer 5'-ACCATTGAAGGTTTGCAACC-3', antisense primer 5'-GGAGGTGCTGTCTGGAGAAA-3' (accession no. X15906); and fibronectin EIII-B sense primer 5'-AGAATAACCACCACCCCCA-3', antisense primer 5'-TGTTAGGACCACGGCGTT-3' (accession no. X15906). DNA amplification was performed with 23 ng of cDNA using the LightCycler technology (Idaho Technology); TGF-beta , fibronectin, and SMA amplification was performed with the LightCycler-FastStart DNA Master SYBR Green I (FastStart DNA Taq Polymerase was activated by preincubation at 95°C for 10 min); beta -actin and CTGF amplification was performed with the LightCycler-DNA Master SYBR Green I (denaturation at 95°C for 30 s). Reactions were cycled 32 times (denaturation at 95°C: TGF-beta 15 s, fibronectin and SMA 10 s, beta -actin and CTGF 1 s; annealing: TGF-beta 63°C, SMA 55°C, beta -actin 58°C, and CTGF and fibronectin 56°C; extension at 72°C: TGF-beta 10 s, SMA 20 s, beta -actin 22 s, fibronectin 25 s, and CTGF 17 s; slopes were 20°C/s). Fluorescence was measured at the end of the extension phase. To confirm the specificity of the amplified products, melting curves were performed at the end of the amplification by cooling the samples at 20°C/s to 73°C (TGF-beta ), 55°C (SMA), 68°C (beta -actin), 56°C (fibronectin), and 66°C (CTGF) and then increasing the temperature to 98°C at 0.1°C/s with fluorescence measurement every 0.1°C. For quantification, standards were generated by purification of PCR products with the QIA quick Gel Extraction Kit, sequenced (Sequiserve, Vaterstetten, Germany), and used as standard in a 10-fold serial dilution after determination of the concentration by absorbance at 260 nm. PCR products were quantified using the LightCycler software.

DNA amplifications of the fibronectin EIII-A and EIII-B splice variants were performed with 12 ng of cDNA using RedTaq DNA Polymerase in a standard protocol, applying one initial step at 94°C for 3 min, 32 cycles at 94°C for 45 s, 56°C for 45 s, and 72°C for 45 s followed by an extension step at 72°C for 5 min using a Mastercycler 5330 (Eppendorf, Hamburg, Germany). No detectable PCR products were present in water controls and in controls amplified without prior reverse transcription. For visualization, PCR products were applied to a 1% agarose gel in 0.5% Tris-borate buffer and stained with GelStar. To confirm that RT-PCR products represent EIII-A+, EIII-A-, EIII-B+, and EIII-B- encoding fragments, PCR products were isolated from agarose gel and sequenced.

Extraction of Total RNA for Northern Blot

PSC were cultured in the presence of 10% FCS in 75-cm2 flasks until confluency. Next, FCS was reduced to 0.1% for 24 h. During another medium change (0.1% FCS), growth factors were added alone and in combination (20 ng/ml TGF-alpha ; 5 ng/ml TGF-beta 1; 50 ng/ml bFGF; 50 ng/ml PDGF-AB). After another 12 h, RNA isolation was performed by the RNeasy Kit according to the manufacturer's instructions. RLT-lysis buffer with 2-mercaptoethanol was added to the cell cultures, and cells were homogenized, pelleted by centrifugation, washed, and isolated by elution. After repeated ethanol precipitations, the total RNA content was calculated by measuring the absorbance at 260 nm.

Northern Blot and Hybridizations

The cDNA probes were labeled with [alpha -32P]dCTP by nick translation using a commercially available Random-Priming Kit as described by the manufacturer. Unincorporated nucleotides were removed by centrifugation of the reaction mixture. Total RNA (30 µg) was denaturated, electrophoretically size fractionated in a 1% agarose gel, and transferred to a Hybond-N membrane. After prehybridization, blots were hybridized overnight with [32P]cDNA probes for the type I and type III collagen, fibronectin, and laminin. Prehybridizations and hybridization were carried out in 6× saline-sodium citrate, 5× Denhardt's solution (50× Denhardt's = 0.02% Ficoll type 400, 0.02% BSA, and 0.02% polyvinylpyrrolidone), 0.5% SDS, 100 µg yeast tRNA/ml, 100 µg/ml sonicated salmon testes/sperm DNA, 10 µg/ml poly(dA/dU) homopolymer, and 50% formamide at 42°C. Filters were washed at high stringency. X-ray films were exposed for ~8-11 days at -70°C. Equal amounts of RNA were loaded on the basis of absorbance at 260 nm, and equivalency of samples was verified by the intensity of 18S rRNA bands. The efficiency of the transfer was judged by viewing the membrane under ultraviolet (302 nm) light.

Western Blot Analysis of PDGF Receptors

PSC were cultured in the presence of 10% FCS or serum starved for 24 h before cell lysis. Cell lysis and Western blot analysis were performed as previously described (49). In brief, equal amounts of protein lysate were run out on a 7.5% SDS-PAGE and blotted on a Hybond C extra membrane. Immunoblot analysis of PDGF-alpha and -beta receptors was performed using specific polyclonal antisera (Santa Cruz Biotechnology, Santa Cruz, CA), an HRP-conjugated anti-rabbit antiserum, and a chemiluminescence-based detection system (Pierce).

Statistical Analysis

The quantitative measurements of fibronectin and BrdU were done in duplicate, and DNA was measured in triplicate. Results are presented as means ± SD of at least three independent experiments, with each condition performed with three cultures. ANOVA (Scheffé's test) was employed to compare different groups. An alpha -level of P < 0.05 was considered to be statistically significant.


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Purity and Morphology of Cultured Rat PSC

After collagenase disaggregation and density gradient centrifugation, 2-6 × 106 fat-storing cells were obtained from rat pancreas. The purity of the isolated cells was assessed in primary culture by illuminated microscopy, phase-contrast microscopy, electron microscopy, vitamin A autofluorescence, and positive staining of vimentin. PSC freshly isolated after collagenase digestion by density gradient centrifugation and cultured cells within the first hours after seeding had a compact spherical appearance (data not shown). There was no morphological difference between cells isolated by density gradient centrifugation from normal or injured pancreas (2 days after cerulein injection). However, cell number and plating efficiency were somewhat higher (30-50%) if PSC were isolated from injured (cerulein) pancreas. During the first 3 days in culture, the cell diameter increased severalfold. The most characteristic structural features in early culture were the perinuclear lipid droplets (Fig. 1A), which decreased in number and size during primary culture and in particular after passage (Fig. 1B). The purity of the isolated PSC as determined by cell morphology (spindle shaped or triangular), the presence of perinuclear fat droplets, and vitamin A autofluorescence, and the expression of the cytofilaments vimentin (data not shown), desmin (data not shown), and SMA (Fig. 1, C and D) were >80% in late primary culture. After the first passage, cell cultures were almost free of contaminating cells. After passage, cell morphology, expression of the cytofilaments, cell growth, and extracellular matrix synthesizing capacity were not different between PSC isolated by the density gradient method from normal and injured pancreas or by the outgrowth method. Passaged cells (1st to 6th passage) always represented activated PSC (myofibroblast phenotype). In passaged cells, expression of vimentin and SMA was almost 100%, and expression of desmin was 20-40%.


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Fig. 1.   Morphology of cultured rat pancreatic stellate cells (PSC). A: phase-contrast microscopy of primary cultured rat PSC. Cells were isolated after collagenase digestion of pancreas from male Wistar rats after cerulein-induced pancreatitis by density gradient centrifugation and were seeded on glass coverslips. After seeding (48 h), phase-contrast micoscopy was performed. Most cells are spindle shaped and contain 6-20 perinuclear fat droplets (original magnification, ×200). B: phase-contrast microscopy of a cultured rat PSC 24 h after passage. The size and number of the fat droplets is reduced. A cell body and long cytoplasmic extensions determine the stellate shape (original magnification, ×200). C: expression of smooth muscle alpha -actin in early primary culture rat PSC. Cells were isolated after collagenase digestion of pancreas from male Wistar rats after cerulein-induced pancreatitis by density gradient centrifugation and were seeded on glass coverslips with a density of 4 × 104 cells/cm2. Later (2 days), cells were fixed with acetone, and indirect immunofluorescence staining using anti-smooth muscle alpha -actin was performed (original magnification, ×200). D: expression of smooth muscle alpha -actin in late primary culture rat PSC. Cells were isolated and seeded as described in C. Later (7 days), cells were fixed, and indirect immunofluorescence staining for smooth muscle alpha -actin was performed (original magnification, ×200). Compared with early culture PSC, all late primary culture PSC show intense actin staining with prominent stress fibers. F, fat droplets; N, nucleus.

Activation of Cultured PSC: Change in Cell Phenotype

Within 4-8 days in primary culture, the cells changed their phenotype from a compact fat-storing morphology to a myofibroblast-like cell. Hereby, the size and number of the fat droplets decreased, and the cells flattened and became stellate-like with spindle-shaped or long cytoplasmic extensions (Fig. 1B). Cells showing the activated phenotype in late primary culture and after passage were characterized by phase-contrast microscopy (Fig. 1B), electron microscopy (remnants of cytoplasmatic fat droplets, prominent endoplasmic reticulum with cisternae, and a large nucleus), and positive SMA staining (Figs. 1D and 2I). As shown in Fig. 2, in parallel to the change in cell morphology, the expression of SMA (Fig. 2, A, E, and I), collagen I (Fig. 2, B, F, and J), collagen III (Fig. 2, C, G, and K), and fibronectin (Fig. 2, D, H, and L) increased during the first 5 days in primary culture. The fluorescence signal was weak in PSC stopped 24 and 72 h after seeding but increased significantly in PSC cultured for 5 days (Fig. 2).


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Fig. 2.   PSC activation during primary culture demonstrated by immunofluorescence microscopy of smooth muscle alpha -actin, collagen types I and III, and fibronectin. Cells were isolated after collagenase digestion of pancreas from normal Wistar rats by density gradient centrifugation and were seeded with a density of 0.5 × 104 cells/cm2 on glass coverslips. Cells were cultured in the presence of 10% FCS. After seeding (1, 3, and 5 days), cultures were stopped, and indirect immunofluorescence stainings using anti-smooth muscle alpha -actin, anti-collagen I and III, and anti-fibronectin were performed (original magnification, ×200). A-D: fixation 24 h after seeding; E-H: fixation 72 h after seeding; I-L: fixation 120 h after seeding; A, E, and I: smooth muscle alpha -actin; B, F, and J: collagen type I; C, G, and K: collagen type III; D, H, and L: fibronectin.

To demonstrate the change in cell phenotype on the mRNA level, PSC were cultured in the presence of 10% FCS and stopped on days 1, 3, 5, and 7 after seeding and day 3 after passage. Total RNA was extracted and reverse transcribed into cDNA. DNA was amplified using the LightCycler technology and specific oligonucleotide primers for fibronectin, SMA, TGF-beta 1, CTGF, and beta -actin. As shown in Fig. 3 in particular, the steady-state levels of the fibronectin and SMA mRNA increased dramatically during the first 7 days in primary culture. During the first 3 days after seeding, TGF-beta 1 mRNA increased 7-fold and during the first 5 days 38-fold (Fig. 3). The increase in CTGF mRNA was lower compared with the mRNA of fibronectin, SMA, TGF-beta 1, and the "housekeeping" beta -actin mRNA. After passage, the expression of SMA mRNA further increased to almost 3,000-fold of the level measured 24 h after seeding (Fig. 3).


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Fig. 3.   PSC activation during primary culture demonstrated by real-time quantitative RT-PCR of fibronectin, smooth muscle (sm) alpha -actin, transforming growth factor (TGF)-beta 1, connective tissue growth factor (CTGF), and beta -actin. Cells were isolated after collagenase digestion of pancreas from normal Wistar rats by density gradient centrifugation and were seeded with a density of 0.5 × 104 cells/cm2 on 6-well culture plates. Cells were cultured in the presence of 10% FCS, and medium was changed 24, 72, and 120 h after seeding and 24 h after passage. To minimize the effect of FCS on the steady-state level of mRNAs, FCS was reduced to 0.1% 12 h before each culture stop. After seeding (3, 5, and 7 days) and 3 days after passage, cultures were stopped, and total RNA was extracted. After determination, the concentration and purity of 570 ng total RNA were reverse transcribed into cDNA. DNA amplification was performed during 32 cycles starting with 23 ng of cDNA. Specific oligonucleotide primers for fibronectin, smooth muscle alpha -actin, TGF-beta 1, CTGF, and beta -actin and the LightCycler technology were used. Fluorescence was measured at the end of the extension phase. To confirm the specificity of the amplified products, melting curves were performed at the end of amplification. For quantification, standards were generated by purification of PCR products isolated from agarose gels and used in 10-fold serial dilution after determination of the concentration by absorbance at 260 nm. PCR products were quantified using the LightCycler software. Values are presented as means of 2 independent experiments and are based on the mRNA steady-state levels of the cultures, which were stopped 24 h after seeding (representing control = 1). p, Passage; 1st pass, culture 72 h after passage. Inset: agarose gel electrophoresis of the PCR products obtained by amplification with the LightCycler. Lane 1, base pair (bp) ladder; lane 2, beta -actin (540 bp); lane 3, TGF-beta 1 (234 bp); lane 4, smooth muscle alpha -actin (293 bp); lane 5, CTGF (432 bp).

The effect of growth factors on PSC activation was studied by performing immunofluorescence microscopy of SMA after addition of growth factors to primary cultured PSC (isolated from normal rat pancreas). To avoid activation by FCS, cells were cultured with reduced (0.1%) FCS. Growth factors were added in appropriate concentrations 24 and 72 h after seeding. As shown in Fig. 4B, positive SMA immunofluorescence is visible in 3-day-old primary cultured PSC only in the presence of TGF-beta 1. At that time point, control cells and TNF-alpha -treated cells are SMA negative. After 5 days, TGF-beta 1-treated PSC show strong SMA immunofluorescence, and also TNF-alpha -stimulated PSC are SMA positive (Fig. 4F). The effects of TGF-beta 1 and TNF-alpha are not additive (data not shown).


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Fig. 4.   Effect of TGF-beta 1 and TNF-alpha on PSC activation during primary culture demonstrated by immunofluorescence microscopy of smooth muscle alpha -actin. Cells were isolated from normal Wistar rats by density gradient centrifugation and were seeded with a density of 0.5 × 104 cells/cm2 on glass coverslips. Cells were cultured in the presence of 0.1% FCS. TGF-beta 1 (2 ng/ml) and TNF-alpha (8 ng/ml) were added 24 and 72 h after seeding. After seeding (3 and 5 days), cultures were stopped, and indirect immunofluorescence stainings using anti-smooth muscle alpha -actin were performed. A-C: fixation 72 h after seeding; D-F: fixation 120 h after seeding; A and D: control (without growth factors); B and E: 2 ng/ml TGF-beta 1; C and F: 8 ng/ml TNF-alpha . Original magnification, ×200.

Because PDGF-AB represented the most important mitogen in activated PSC (see below) but not in PSC with the fat-storing phenotype (data not shown), we studied the expression of PDGF alpha - and beta -receptors by Western blot in cultured PSC. As shown in Fig. 5, late primary cultured rat PSC abundantly express both PDGF receptors, the alpha - and, in particular, the beta -type. The presence of serum upregulated both receptor types (Fig. 5, A and B). PDGF receptors were not expressed in early culture (24 and 72 h after seeding) PSC (24 and 72 h after seeding). ZnR5 cells, known to express only PDGF beta -receptors, were used as control (Fig. 5).


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Fig. 5.   Expression of the platelet-derived growth factor (PDGF) alpha -receptor (A) and of the PDGF beta -receptor (B) in late primary cultured PSC. Cells were isolated and cultured in 10% FCS as described in the legend for Fig. 3. Before cell lysis (24 h), part of the cells was serum starved. Cultures were stopped 7 days after seeding. PSC constitutively express PDGF alpha -receptor protein (A) and PDGF beta -receptor protein (B). Both the glycosylated protein and the precursor molecule can be detected. The expression level is elevated under high-serum conditions. ZnR5 cells expressing the human PDGF beta -receptor are used as a control. IB, immunoblot antibody.

Effects of Growth Factors on Cell Proliferation

We explored the effects of the polypeptides PDGF-AB, bFGF, TGF-beta 1, TGF-alpha , TNF-alpha , IGF-I, and IGF-II alone and combinations of these polypeptides on BrdU incorporation into DNA of activated PSC (cells in 2nd or 3rd passage). PDGF-AB and bFGF (both 50 ng/ml) stimulated PSC proliferation to 4.37 ± 0.49- and 2.96 ± 0.39-fold of control, respectively (Fig. 6A). Control PSC were cultured in the presence of 0.5% FCS. No significant stimulation of BrdU incorporation was obtained after addition of TGF-beta 1, TGF-alpha , TNF-alpha , IGF-I, and IGF-II (Fig. 6A). The effects of PDGF-AB and bFGF were not additive if both polypeptides were added simultaneously (Fig. 6B). All growth factor combinations with PDGF-AB significantly stimulated BrdU incorporation (Fig. 6B). If TGF-beta 1 was present in growth factor combinations, the mitogenic effect was reduced by 30-50% (Fig. 6B).


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Fig. 6.   Effect of growth factors on PSC proliferation. Cells were isolated by the outgrowth method using pancreas tissue of rats with cerulein pancreatitis. After reaching confluence, cells were trypsinized and subcultured in a 1:3 ratio. Two days after passage, growth factors [TGF-beta 1 5 ng/ml, TGF-alpha 20 ng/ml, basic fibroblast growth factor (bFGF) 50 ng/ml, PDGF-AB 50 ng/ml, insulin-like growth factor (IGF)-I 50 ng/ml, IGF-II 50 ng/ml, tumor necrosis factor (TNF)-alpha 8 ng/ml medium] were added alone (A) or in combination (B) to PSC (after 2nd or 3rd passage) in the presence of 0.5% FCS. Later (6 h), bromodeoxyuridine (BrdU; final concentration 5 × 10-5 M) was added for another 18 h. Thereafter, cultures were stopped, and BrdU incorporation was measured as described in MATERIALS AND METHODS by time-resolved immunofluorescence using an Europium chelate. The results are expressed as a fraction of control (cells grown in DMEM with 0.5% FCS) and represent the means ± SD of 3 experiments (each condition performed in triplicate wells). *Statistically significant difference (P < 0.05) compared with control.

Effects of Growth Factors on the Synthesis of Extracellular Matrix

The effects of growth factors on extracellular matrix synthesis of cultured PSC were demonstrated by measuring the fibronectin concentration in cell culture supernatants (Fig. 7), by performing immunofluorescence stainings of cell-associated collagen types I and III and fibronectin (Fig. 8), and by performing a Northern blot to detect the steady-state levels of the mRNA of collagen I, collagen III, fibronectin, and laminin (Fig. 9). bFGF (5, 20, and 50 ng/ml), TGF-beta 1 (0.5, 2, and 5 ng/ml), PDGF-AB (5, 20, and 50 ng/ml), and to a lesser amount TGF-alpha (5, 20, and 50 ng/ml) stimulated fibronectin synthesis of cultured PSC (Fig. 7). Neither TNF-alpha nor IGF-I or IGF-II stimulated fibronectin synthesis of cultured rat PSC (Fig. 7). Among the different growth factor combinations, only bFGF with PDGF-AB and bFGF with PDGF-AB and TGF-alpha significantly stimulated fibronectin synthesis (data not shown).


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Fig. 7.   Effect of polypeptide growth factors on fibronectin synthesis of cultured rat PSC. After passage (2 days), growth factors (TGF-beta 1 0.5, 2, and 5 ng/ml; TGF-alpha 5, 20, and 50 ng/ml; PDGF-AB 5, 20, and 50 ng/ml; bFGF 5, 20, and 50 ng/ml; TNF-alpha 3, 8, and 20 ng/ml; IGF-I 5, 20, and 50 ng/ml; and IGF-II 5, 20, and 50 ng/ml) were added to PSC (after 2nd-4th passage) grown in DMEM with 0.5% FCS. After 24 h, cultures were stopped, and fibronectin was measured in PSC supernatants; DNA was measured in the cell layer. Values are expressed as means ± SE of 3 independent experiments, with each condition performed in triplicate culture wells.



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Fig. 8.   Fluorescence micrographs showing the immunoreactivity of collagen type I (A, D, G, J, and M), collagen type III (B, E, H, K, and N), and fibronectin (C, F, I, L, and O) in cultured rat PSC stimulated with TGF-beta 1, PDGF-AB, TGF-alpha , and bFGF. Cultured rat PSC (4th passage) grown on glass coverslips were stimulated for 24 h with TGF-beta 1, PDGF-AB, TGF-alpha , and bFGF. Thereafter, cultures were washed and fixed in acetone, and immunostaining was performed. A-C: control 0.5% FCS; D-F: 5 ng/ml TGF-beta 1; G-I: 50 ng/ml PDGF-AB; J-L: 20 ng/ml TGF-alpha ; M-O: 50 ng/ml bFGF. To compare staining intensities, all micrographs were taken using the same exposure time. Original magnification, ×400.



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Fig. 9.   Northern blot hybridization of collagen I (alpha 1), collagen III (alpha 1), fibronectin, laminin, and 18S rRNA. After passage (2 days), growth factors (TGF-beta 1 5 ng/ml, TGF-alpha 20 ng/ml, bFGF 50 ng/ml, PDGF-AB 50 ng/ml, IGF-I 50 ng/ml, IGF-II 50 ng/ml, TNF-alpha 8 ng/ml medium) were added alone or in combination to PSC (after 2nd or 3rd passage) in the presence of 0.5% FCS. Later (12 h), cultures were stopped, and RNA was isolated. RNA (30 µg/lane) was separated by agarose gel electrophoresis and was transferred to Hybond-N membranes. Ethidium bromide staining of the agarose gels was used to verify equal loading and blotting of total RNA (data not shown). Hybridizations were performed by standard procedures using 0.5 × 106 counts · min-1 · ml-1 labeled cDNA. Exposure to X-ray films was done at -70°C for 7-10 days. Lane 1: 0.1% FCS (control); lane 2: 5 ng/ml TGF-beta 1; lane 3: 20 ng/ml TGF-alpha ; lane 4: 50 ng/ml PDGF-AB; lane 5: 50 ng/ml bFGF; lane 6: TGF-beta 1 with bFGF; lane 7: TGF-beta 1, TGF-alpha , PDGF, and bFGF; lane 8: TGF-beta 1, TGF-alpha , and bFGF; lane 9: 10% FCS.

To demonstrate the effects of growth factors on cell-associated collagens and fibronectin, cultured rat PSC were incubated for 24 h with 0.5% FCS (control) or 50 ng/ml bFGF, 5 ng/ml TGF-beta 1, 50 ng/ml PDGF-AB, and 20 ng/ml TGF-alpha together with 0.5% FCS. Thereafter, cultures were fixed, and immunostainings were performed. As shown in Fig. 8, intensive staining patterns of collagen type I (D, G, J, and M), collagen type III (E, H, K, and N), and fibronectin (F, I, L, and O) were observed in cultures receiving TGF-beta 1 (D-F), PDGF (G-I), TGF-alpha (J-L), and bFGF (M-O). The most intensive immunostaining of all extracellular matrix components was observed after addition of bFGF (Fig. 8, M-O). Collagen type I immunofluorescence was detected intracellularly and in extracellular fibrils varying in diameter and intensity. Collagen type III was detected predominantly intracellularly with the highest intensity in the perinuclear region. Fibronectin was located in extracellular fibrils varying in density. Immunofluorescence of collagens and fibronectin in TNF-alpha - and IGF-treated cultures was not different from control (data not shown). Addition of PDGF also increased cell density compared with control (Fig. 8, G-I).

As shown in Fig. 9, the steady-state levels of the mRNA of collagen type I and III and fibronectin were increased by bFGF and growth factor combinations, including bFGF. The steady-state level of laminin was not influenced by the different growth factors.

To analyze different splice sites of fibronectin (EIII-A and EIII-B) and their level of expression, primary cultured PSC were stopped 1, 3, 5, and 7 days after seeding and 3 and 5 days after the first passage. As shown in Fig. 10, amplification of the fragment including the EIII-B site resulted in the appearance of two products (one product of 629 bp including the EIII-B exon and the other of 358 bp lacking the EIII-B exon), indicating that both EIII-B fibronectin mRNA forms are present, whereas the EIII-B+ form is weakly expressed (Fig. 10). The amplification of the region that includes the EIII-A site also gave two products (one of 267 bp in which the EIII-A exon is excluded and one of 536 bp in which the EIII-A exon is included). Compared with EIII-B, the expression of EIII-A-mRNA is pronounced at days 5 and 7 in primary culture and day 3 after passage. Both splice variants could not be detected at days 1 and 3 of primary culture (Fig. 10). As shown by real-time quantitative RT-PCR at the same time points, the expression levels of beta -actin and fibronectin mRNA were low (Fig. 3).


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Fig. 10.   Demonstration of fibronectin splice variants by RT-PCR of EIII-A and EIII-B. An equal amount of RNA (570 ng), isolated from cultured rat PSC, was reverse transcribed and amplified during 32 cycles. Lanes 1-6: RNA isolated at day 1 (lane 1), day 3 (lane 2), day 5 (lane 3), and day 7 (lane 4) of primary culture and day 3 (lane 5) and day 5 (lane 6) after first passage. Lane A represents the bp ladder. EIII-A, left side of bp ladder; EIII-B, right side of bp ladder.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In normal pancreas, low numbers of PSC with the fat-storing phenotype are located in periacinar and interlobular regions. However, in pancreas fibrosis, activated PSC expressing SMA have been found in high numbers associated with connective tissue (8, 26, 40). Interestingly, activated PSC derived from human and rat pancreas synthesize and secrete the same extracellular matrix components, namely collagen types I and III and fibronectin (8), which are found in pancreas fibrosis (31). Furthermore, immunostaining of SMA combined with in situ hybridization for procollagen mRNA identified activated PSC as a principal source of collagen synthesis in experimental chronic pancreatitis (26). Because the first step in pancreas fibrogenesis is PSC activation (change of the cell phenotype), the aim of the present study was to identify polypeptide fibrogenic mediators stimulating PSC activation. Furthermore, because proliferation and extracellular matrix synthesis of activated PSC are preconditions of fibrogenesis, we were interested in the identification of fibrogenic mediators stimulating proliferation and matrix synthesis of activated PSC.

To study the transition from the resting fat-storing to the highly active myofibroblast-like phenotype, PSC representing fat-storing cells have to be isolated and cultured. To study the effects of growth factors on proliferation and matrix synthesis of activated PSC, the myofibroblast-like phenotype has to be used. An important prerequisite to perform this study was a high purity of the isolated "fat-storing" PSC and high numbers of activated "myofibroblast-like" PSC. To isolate PSC we used two different methods. To obtain cells with the inactivated fat-storing phenotype, cells were isolated by density gradient centrifugation after collagenase digestion of pancreas from normal Wistar rats (200-300 g) or from rats 2 days after cerulein injection. To get higher numbers of cells, the outgrowth method was used with the pancreas of cerulein-treated rats. The outgrowth method, which is not applicable with normal pancreas, provides PSC with the activated (myofibroblast-like) phenotype. In late primary culture and after passage, no difference regarding cell morphology, cell growth, expression of cytofilaments, and matrix synthesis could be detected between cells isolated by the density gradient method and the outgrowth method. Research groups interested primarily in liver fibrosis frequently use the outgrowth technique to isolate activated HSC from fibrotic livers (10).

The purity of our PSC preparations isolated by density gradient was >80%, and after the first passage our PSC cultures were almost free of contaminating acinar cells, ductal cells, fibroblasts, and endothelial cells. Almost 100% of the PSC in late primary culture and subsequent passages were SMA positive. During primary culture and, in particular, after passage, we observed the characteristic morphological changes associated with the change in the cell phenotype (8). These changes include the increase in cell diameter, the reduction of the number and size of perinuclear retinoid-containing fat droplets, and an increased expression of desmin and SMA. As shown by immunofluorescence stainings and real-time quantitative RT-PCR in parallel with PSC activation, the expression of extracellular matrix proteins increased, and late primary cultured PSC (myofibroblast-like phenotype) and secondary cultured PSC had a high capacity to synthesize and secrete extracellular matrix. Interestingly, the increase in the mRNA of TGF-beta 1 during the first days in primary culture was more pronounced compared with the mRNA of CTGF. As shown very recently by Vogelmann et al. (48), TGF-beta 1 alone seems to be sufficient to induce pancreas fibrosis in vivo, whereas CTGF and bFGF appear to represent cofactors at later stages. CTGF represents a downstream mediator of TGF-beta 1, as shown by Grotendorst (25) in fibroblasts. An upregulation of CTGF mRNA expression was recently shown in human chronic pancreatitis (15) and pancreas carcinoma (51). Our result that TGF-beta 1 accelerates the change in the cell phenotype is compatible with the in vivo data indicating that in particular TGF-beta 1 is responsible for the first step in fibrogenesis, namely PSC activation. Probably also in PSC, CTGF represents a downstream mediator of TGF-beta 1 action on matrix synthesis. However, it seems unlikely that the effect of TGF-beta 1 on PSC activation is mediated through upregulation of CTGF.

The question about the source of TGF-beta in pancreas injury has been studied earlier by our group (23). We have shown that, in rat cerulein pancreatitis, TGF-beta 1 protein increased earlier than TGF-beta mRNA in pancreas tissue, suggesting that platelets might be the source of the early TGF-beta 1 protein increase (23). Thereafter TGF-beta 1 was released by inflammatory cells, stromal cells, and acinar cells (23). To inhibit the TGF-beta effects during regeneration from cerulein pancreatitis in rats, Menke et al. (38) injected TGF-beta 1-neutralizing antibodies and could show that, by this approach, collagen type I and III protein and mRNA decreased significantly compared with control (without anti-TGF-beta 1). Furthermore, anti-TGF-beta 1 inhibited the usually observed rise of the steady-state levels of TGF-beta 1 mRNA and TGF-beta 2 mRNA at the second day after cerulein infusion (38), suggesting that the early TGF-beta 1 release by platelets induces the TGF-beta synthesis.

From the present and previously obtained data, we suggest the following pathogenic sequence of events leading to the conversion of resting PSC to highly activated myofibroblast-like cells in vivo. Independent of the responsible pathomechanism, acinar cell injury causes aggregation of platelets in pancreatic capillaries (34) and extravasation of mononuclear cells into the damaged tissue. Emmerich et al. (17) described a three- to fourfold increased number of infiltrating macrophages in chronic panreatitis. Aggregating platelets and leucocytes release cytokines and growth factors, e.g., interleukin (IL)-1, IL-6, TNF-alpha , and TGF-beta (4, 5, 37). Hereby, a basic level of TNF-alpha and TGF-beta is obtained, which is sufficient to trigger activation of PSC. As a consequence of PSC activation, PDGF receptors are expressed, cell proliferation accelerates, matrix synthesis increases, and the cells start to express TGF-beta 1. Preliminary data suggest that autocrine stimulatory loops via TGF-beta 1 might enhance extracellular matrix synthesis (7). Addition of TGF-beta 1-neutralizing antibodies and a TGF-beta 1 antisense oligonucleotide significantly reduced extracellular matrix synthesis of activated PSC (7).

To identify the mediators stimulating activated PSC, we added the growth factors PDGF-AB, bFGF, TGF-beta 1, TGF-alpha , TNF-alpha , IGF-I, and IGF-II to secondary cultured PSC and measured cell proliferation and extracellular matrix synthesis. Cell proliferation was stimulated by PDGF-AB and bFGF. No significant mitogenic effect was associated with TGF-beta 1, TGF-alpha , TNF-alpha , IGF-I, and IGF-II. Our data obtained after addition of PDGF are in accordance with earlier reports showing that PDGF stimulated proliferation of cultured PSC (2) and "fibroblast-like cells" derived from cultured human pancreatic acini (42). Two reports indicate an association of PDGF with pancreas fibrosis. Slater and coworkers (44) demonstrated an increased expression of PDGF-BB and its receptor in chronic pancreatitis, and Haber et al. (26) have shown recently that in areas of fibrosis the PDGF-beta receptor is closely associated with desmin staining, suggesting that PSC express the PDGF beta -receptor. According to our data also, the PDGF alpha -receptor is expressed by cultured PSC, although the PDGF beta -receptor was stronger in the Western blot.

We identified bFGF, TGF-beta 1, and PDGF as fibrogenic mediators stimulating the synthesis of collagen types I and III and fibronectin. Interestingly, with the use of primary cultured human "fibroblast-like cells" derived from cultured pancreatic acini (42) and cultured human PSC derived from fibrotic pancreas (8), TGF-beta 1 was identified to represent the most potent fibrogenic mediator stimulating matrix synthesis. In contrast to TGF-alpha , bFGF, and PDGF, only TGF-beta 1 stimulated fibronectin synthesis of cultured human PSC in the presence of 1-10% FCS (8). Interestingly, bFGF added to activated mice PSC isolated from fibrotic pancreas of transgenic mice overexpressing TGF-beta 1 stimulated the synthesis of collagen type I and fibronectin, whereas TGF-beta 1 only stimulated collagen type I (48). Obviously, species differences contribute to the discrepancy that fibronectin synthesis is stimulated in human pancreas predominantly by TGF-beta 1 and in rodents by bFGF. However, the central role of TGF-beta in experimental and human pancreas fibrogenesis is well documented (for review, see Refs. 22 and 39). By applying a pancreatitis model in the rat, Kimura et al. (32) demonstrated by immunohistochemistry an association between the deposition of extracellular matrix, TGF-beta , and inflammatory cells. Furthermore, increased TGF-beta expression was observed within acinar cells adjacent to areas of fibrosis, suggesting that acinar cells represent a significant cellular source of TGF-beta in human and rat pancreas fibrogenesis (26). As mentioned above, other sources for TGF-beta and PDGF are aggregating platelets and inflammatory cells, e.g., macrophages. Recently, we have shown by preincubation of platelet lysates with neutralizing antibodies to TGF-beta 1 and PDGF that TGF-beta 1 represents the main mediator stimulating matrix synthesis and PDGF as the most important mitogen released by aggregating platelets (34). Recently obtained data suggest that initially released TGF-beta 1 could induce its own de novo synthesis in PSC, hereby propagating self perpetuation of pancreas fibrogenesis by an autocrine stimulatory loop (30). Autocrine stimulation of collagen type I and fibronectin synthesis of cultured PSC could be reduced by addition of TGF-beta 1-neutralizing antibodies and a triplex-forming oligonucleotide binding to the promoter region of the TGF-beta 1 gene (7).

It is unlikely that bFGF is also involved in autocrine stimulation of matrix synthesis of PSC. Friess and coworkers (20) have shown years ago that bFGF and its receptor are overexpressed in pancreas tissue of patients with chronic pancreatitis. With the use of in situ hybridization and immunohistochemistry, bFGF overexpression was localized in the cytoplasm of ductal and acinar cells and absent in connective tissue and stromal fibroblasts (20). Taking these and our data into account, we suggest that, in fibrogenesis associated with chronic pancreatitis, ductal cells and acinar cells synthesize and release bFGF, hereby stimulating extracellular matrix synthesis of PSC in a paracrine way. bFGF expression is enhanced in exocrine-type cells not only in chronic pancreatitis but also during regeneration after acute pancreatitis bFGF expression is enhanced in exocrine-type cells (16), suggesting that bFGF might also be involved in the process of pancreas regeneration during recovery from acute pancreatitis.

Furthermore, our data demonstrate an enormous increase of total fibronectin expression during primary culture of PSC and the presence of the embryonic fibronectins, particularly the EIII-A+ mRNA in activated PSC. The expression of the various forms of fibronectin mRNA in vivo is regulated in a tissue- and cell-specific manner during development and in tissue repair and disease (18, 35). During early embryogenesis, most fibronectin mRNA is EIII-A+ and EIII-B+, whereas later in development these exons are excluded in a cell- and tissue-specific pattern. Caputi et al. (12) have shown a remarkable increase of the EIII-A+ form in a rat regenerating liver model. A reappearance of EIII-A+ and EIII-B+ mRNA in adult rat sciatic nerve after a crush lesion suggests that reexpression of embryonic fibronectins may be an important part of a successful repair (48). The EIII-A+ mRNA is of particular interest because a recombinant EIII-A segment has been reported to transform rat lipocytes into myofibroblasts (29).

In summary, our data identified TGF-beta 1 and TGF-alpha as mediators stimulating PSC activation (change in the cell phenotype) and bFGF, TGF-beta 1, and PDGF as fibrogenic mediators stimulating extracellular matrix synthesis of activated rat PSC. In addition, PDGF and bFGF accelerate proliferation of PSC. The origin of these growth factors are platelets (TGF-beta and PDGF) aggregating in injured sites of the pancreas and inflammatory cells (TGF-beta and PDGF) invading the injured pancreas, ductal cells, and acinar cells (bFGF). Furthermore, at least TGF-beta 1 seems to be involved in autocrine stimulation of PSC (7, 30).


    ACKNOWLEDGEMENTS

We thank Martina de Groot and Martina Adam-Jaeger for expert technical assistance. Western blots to demonstrate PDGF receptors were performed by N. Meyer.


    FOOTNOTES

* E. Schneider and A. Schmid-Kotsas contributed equally to this work.

This work was supported by grants from Bausteinförderung University of Ulm (P.347 to M. G. Bachem), Deutsche Forschungsgemeinschaft (SFB 518, Project A7 to M. G. Bachem), and German-Israeli-Foundation (to J. Waltenberger).

Address for reprint requests and other correspondence: M. G. Bachem, Universität Ulm-Klinikum-Institut für Klinische Chemie, Bereichslabor Michelsberg, 89070 Ulm Germany (E-mail: max.bachem{at}medizin.uni-ulm.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 August 2000; accepted in final form 23 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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