Nonoxidative ethanol metabolites alter extracellular matrix protein content in rat pancreas

GASTROENTEROLOGY 2003;125:1845–1859

Nonoxidative Ethanol Metabolites Alter Extracellular Matrix Protein Content in Rat Pancreas AURELIA LUGEA, ILYA GUKOVSKY, ANNA S. GUKOVSKAYA, and STEPHEN J. PANDOL University of Southern California-University of California, Los Angeles, Research Center for Alcoholic Liver and Pancreatic Diseases, Veterans Affairs Greater Los Angeles Healthcare System, and University of California, Los Angeles, California

Background & Aims: The mechanisms involved in ethanol-induced pancreas fibrosis are poorly understood. Here we show that fatty acid ethyl esters (FAEEs), nonoxidative ethanol metabolites, increase extracellular matrix (ECM) protein levels in pancreas. Methods: Rat pancreatic acini were incubated for 1– 4 hours with FAEEs or acetaldehyde. In another set of experiments, rats received an intravenous infusion of FAEEs for 6 hours. Collagens were assessed by a hydroxyproline assay. Laminin and fibronectin were analyzed by Western blotting. Gene expression of ECM proteins was measured by conventional and real-time reverse-transcription polymerase chain reaction (RT-PCR). Matrix metalloproteinase (MMP), plasmin, and urokinase-type plasminogen activator (uPA) activities were determined by zymography and fluorogenic assays. Results: FAEEs increased collagen, laminin, and fibronectin levels in pancreatic acini without affecting messenger RNA (mRNA) expression for these proteins. Actinomycin D, a transcriptional inhibitor, did not block the increase in ECM proteins induced by FAEEs. FAEEs reduced the activity of the serine protease, plasmin, and that of the uPA. Consistent with these results, the serine protease inhibitor aprotinin reproduced the effects of FAEEs and prevented the further increase in ECM proteins induced by FAEEs. In vivo administration of FAEEs reduced plasmin and uPA activities and increased ECM protein levels in pancreas. Acetaldehyde had minor effects on ECM protein levels and did not affect plasmin activity. Conclusions: FAEEs increase ECM protein levels in pancreas. The results suggest that this effect is caused primarily by an inhibition in ECM degradation via serine proteases including the plasminogen system.

ibrosis is one of the main features of alcohol-induced chronic pancreatitis. Fibrosis is caused by abnormal accumulation of extracellular matrix (ECM) in basement membranes as well as interlobular and periacinar areas.1 The abnormal ECM production in fibrosis comprises an excess of normal components such as fibronectin, laminin, type IV collagen, and proteoglycans, as well as an accumulation of proteins that are not found in normal ECM, such as collagens type I and III.2 Fibrosis is a

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dynamic process in which both altered matrix degradation and abnormal ECM protein synthesis play major roles.3,4 Two main ECM-degrading systems are known to participate in tissue remodeling: matrix metalloproteinases (MMPs)5,6 and serine proteases from the plasminogen (Plg) system.7,8 Both systems have been suggested to be crucial modulators in chronic pancreatitis.9,10 The molecular mechanisms resulting in alcoholinduced fibrosis in pancreas remain unknown. Both parenchymal and stellate cells may participate in matrix remodeling through the regulation of ECM production and matrix degradation. It now generally is accepted that pancreatic stellate cells (PSCs) are the major ECM-producing cells in pancreas.11–13 On activation by different stimuli, such as growth factors, cytokines, ethanol, and oxidative stress, PSCs produce large amounts of ECM proteins.14 –16 Active PSCs also express components of the ECM degrading systems.10,17 Although acinar cells have the capacity to synthesize ECM proteins and expressed ECM-degrading proteases,18,19 little is known about their role in pancreatic fibrosis. Ethanol and its metabolites previously have been proposed as inducers of pancreatic fibrosis.20,21 A recent study from this laboratory showed that rat pancreatic acinar cells metabolize ethanol through equally important oxidative and nonoxidative pathways.22 In the oxidative pathway ethanol is metabolized to acetaldehyde mainly through the action of alcohol dehydrogenase. The nonoxidative pathway leads to the formation of fatty acid ethyl esters (FAEEs) by enzymatic esterification of ethanol and fatty acids. FAEEs have been shown to accumulate in humans after ethanol ingestion,23 with the panAbbreviations used in this paper: ␣-SMA, ␣ smooth muscle actin; ARP, acidic ribosomal phosphoprotein P0 gene; CT, threshold cycle; DMSO, dimethyl sulfoxide; ECM, extracellular matrix; FAEEs, fatty acid ethyl esters; MMPs, matrix metalloproteinases; PLC, phospholipase C; Plg, plasminogen; PSC, pancreatic stellate cell; RT-PCR, reverse-transcription polymerase chain reaction; uPA, urokinase-type plasminogen activator. © 2003 by the American Gastroenterological Association 0016-5085/03/$30.00 doi:10.1053/j.gastro.2003.09.021

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creas showing high levels of FAEE synthase activity.24,25 Several studies have shown the toxicity of both acetaldehyde and FAEEs to cells,26,27 although the role of these ethanol metabolites in fibrogenesis is unclear. Acetaldehyde induces activation and production of collagen by fibroblasts and stellate cells.15,28,29 Recent studies showed the toxicity of FAEEs in pancreas,30,31 but their role in pancreatic fibrosis remains unknown. The aim of the present study was to examine the fibrogenic responses to ethanol metabolites in pancreatic acinar cells. We therefore determined the effects of FAEEs and acetaldehyde on major ECM protein levels; collagens, laminin, and fibronectin; and on the activities of ECM-degrading proteases in dispersed rat pancreatic acini and in the pancreas after intravenous administration of FAEEs. We further compared the ability of ethanol metabolites to alter ECM content in pancreatic stellate cells and pancreatic acini.

Materials and Methods Cell Isolation and Culture Dispersed pancreatic acini were isolated from male Sprague–Dawley rats (75–100 g, Harlan, Madison, WI) by collagenase digestion. Briefly, the pancreas was digested with chromatographically purified collagenase (CLPSA) grade collagenase (Worthington Biochemicals, Freehold, NJ), and the resulting preparation of acini was suspended in 199 medium supplemented with antibiotics (100 U/mL penicillin and 100 ␮g/mL streptomycin). Acini were seeded in 60-mm culture dishes at an approximate density of 1 ⫻ 105 cells/cm2 and incubated for 1– 4 hours at 37°C in a 5% CO2-air humidified atmosphere. PSCs were isolated from dispersed acini by outgrowth method. Briefly, pancreatic acini isolated from one animal as described earlier were placed in 2 mL phosphate-buffered saline. One aliquot of the acini suspension (200 ␮L) was seeded into 75 cm2 culture flasks in 10 mL of Dulbecco’s modified Eagle medium/Ham’s F12 medium (1:1) containing 10% fetal bovine serum, 4 mmol/L glutamine, 0.25 ␮g/mL amphotericin B, and antibiotics (100 U/mL penicillin and 100 ␮g/mL streptomycin). Acini were cultured at 37°C in a 5% CO2-air humidified atmosphere for several days. The culture medium was replaced every day during the next 4 days. After 5 days of primary culture, PSCs were subcultured by trypsinization and replated in 6-well plates at a seeding density of 106 cells/well. All media and supplements were from Gibco BRL (Rockville, MD).

Ethanol Metabolite Incubation Studies ECM protein content was studied in pancreatic acini or cultured PSCs after 1– 4 hours of incubation with FAEEs or acetaldehyde. FAEEs consisted of an equimolecular mixture of ethyl oleate, ethyl palmitate, and ethyl linoleate suspended in

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dimethyl sulfoxide (DMSO) or reconstituted into liposomes. Liposomes were prepared using palmitoyl-oleyl-phosphatidylcholine and palmitoyl-oleyl-phosphatidylglycerol as core components as described earlier.32 Immediately after preparation, FAEEs were added to the incubation medium at a final concentration of 0.5, 1, or 3 mmol/L. Acetaldehyde was added directly to the incubation medium at a final concentration of 0.2 and 1 mmol/L. Pancreatic acini treated with DMSO or empty liposomes served as controls. At the end of the incubation period, cells were harvested and suspended in the appropriate buffer for the assay to be performed.

In Vivo Studies To determine the effect of FAEE administration on ECM content in pancreas in vivo, we used male Sprague– Dawley rats weighing 250 –300 g (Harlan, Madison, WI). Animals were housed under controlled conditions of temperature, humidity, and illumination and maintained on standard rodent chow with free access to tap water. All procedures were approved by the Animal Research Committees of the Veterans Affairs Greater Los Angeles Healthcare System (VAGLAHS) and the University of California. Animals were randomly divided into 2 groups of 6 animals each and subjected to the following procedure: under urethane anesthesia (1.25 g/kg, intraperitoneal, Sigma Chemical, St. Louis, MO), a P-50 catheter was inserted into the left jugular vein for FAEE administration. Afterward, the animals received a continuous intravenous infusion of 0.3 mmol/kg/h FAEEs reconstituted into liposomes (FAEE-treated group) or empty liposomes (control group) for 6 hours. Then, animals were killed and pancreas was collected for analysis of ECM protein content and ECMdegrading protease activity. FAEE dose was estimated as an approximation to previous effective doses in vitro.

Collagen Determination Total collagen was assessed by measuring hydroxyproline content as described earlier.33 Cells were hydrolyzed in 6 mol/L HCl at 110°C for 16 hours. After neutralization, hydroxyproline was oxidized for 20 minutes by adding 1 mL of chloramine T dissolved in sodium-citrate buffer (pH 6.0) and 30% methyl cellosolve (Sigma Chemical). The reaction was stopped with 3 mmol/L perchloric acid, and 1 mL of Ehrlich’s reagent (ICN Biochemicals, Aurora, OH) was added to the samples. Samples were incubated at 60°C for 20 minutes and the absorbance was measured at 550 nm. Hydroxyproline values were expressed as ␮g/mg of total protein or total DNA. New collagen synthesis in PSCs was determined by measuring the incorporation of [14C]-proline into collagenasesensitive proteins.34 After 1 hour of incubation in proline-free medium, PSCs between passages 1–3 were labeled for 16 hours with 2 ␮Ci/mL [14C]-proline (ICN Biochemicals) in 0.1% fetal bovine serum culture medium containing 100 ␮g/mL ascorbic acid and 100 ␮g/mL ␤-amino-proprionitrile. Acetaldehyde and FAEEs were added 3 hours before the end of the incubation period. Cells then were harvested by trypsinization, pooled with the medium, and sonicated. Proteins were precip-

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itated with 10% trichloracetic acid and the pellet was solubilized with 0.2 mol/L NaOH. After neutralization, samples were divided into 2 equal aliquots. One aliquot was incubated with 25 ␮g/mL purified collagenase (CLPSA grade) for 2 hours at 37°C, and the other one was treated with vehicle. Collagenase-resistant proteins were separated from the released collagen peptides by trichloracetic acid precipitation. Radioactivity in the trichloracetic acid–soluble fraction was measured in a liquid scintillation counter, and newly synthesized collagen was determined by the difference between the radioactivities found in the treated and nontreated collagenase aliquots. Collagen synthesis was expressed as cpm/␮g of total DNA.

Immunoblot Analysis Cells were lysed in RIPA buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and a freshly added mixture of protease inhibitors (5 ␮g/mL each of aprotinin, pepstatin, leupeptin, antipain, and chymostatin). Cellular protein extracts were resolved by electrophoresis on 6% (for immunoblot analysis of high molecular weight proteins such as laminin, fibronectin, and phospholipase C ␥-1) or 4%–12% gradient (immunoblot analysis of urokinase and MMP-2) Trisglycine gels (Invitrogen, Carlsbad, CA) and then transferred to nitrocellulose membranes. After blocking nonspecific binding with 5% nonfat dried milk, the membranes were incubated with the following primary antibodies: rabbit anti-rat laminin, fibronectin, desmin, and ␣ smooth muscle actin (␣-SMA; Sigma Chemical) and rabbit anti-mouse MMP-2 (Santa Cruz BioTechnology, Santa Cruz, CA). Mouse anti-rat PLC ␥1 (Santa Cruz BioTechnology Inc.) and mouse anti-rat tubulin antibodies (Neomarkers Inc., Fremont, CA) were used to normalize protein levels on 6% and 4%–12% gradient, respectively. Afterward, membranes were incubated with horseradish peroxidase– conjugated specific secondary antibodies. Immunoreactive bands were visualized on Kodak XAR5 film (Eastman Kodak Company, Rochester, NY) using chemiluminescence detection reagents (Pierce, Rockford, IL). Optical densitometry reading of the blots was performed using Scion imaging software (Scion Corporation, Frederick, MD).

RNA Isolation and Quantification of Gene Expression We first analyzed messenger RNA (mRNA) expression for ECM proteins by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) as described earlier.35 Briefly, total RNA was isolated from pancreatic acini with TRI reagent (Molecular Research Center, Cincinnati, OH), reversetranscribed with the SuperScript II kit (Invitrogen), and subjected to PCR using rat gene–specific, intron-spanning primers. The PCR primers were as follows: collagen ␣1 type I, forward primer, nucleotide positions 4375– 4394, and reverse, 4565– 4587, in the GenBank complementary DNA (cDNA) sequence Z78279; collagen ␣1 type III, forward, nucleotide positions 1199 –1221, and reverse, 1598 –1618, in the GenBank X70369; laminin ␥2, forward, nucleotide positions

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3–24, and reverse, 267–286, in the GenBank Y08884. The primer sequences for the housekeeping acidic ribosomal phosphoprotein P0 (ARP) gene have been described previously. Thirty-five target sequences were amplified at 56°C using the same amount of cDNA for all primer sets. Negative controls were performed by omitting the RT step or cDNA template from the PCR amplification. The cycle number was adjusted between 22 and 31 cycles to yield visible products within the linear amplification range. Resulting RT-PCR products were run on agarose gel and visualized by staining with ethidium bromide. We next quantified collagen mRNA expression by real-time PCR TaqMan assay36 using the ABI Prism 7700 Sequence Detection System and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA). The sequences of the primers used as well as the double-labeled FAM/TAMRA and VIC/TAMRA TaqMan probes for collagen ␣1(I), collagen ␣1(III), and ARP genes are available on request. Because preliminary experiments showed amplification efficiencies of nearly 1.0 for all 3 genes under study, mRNA quantification analysis was performed with the comparative ⌬⌬CT method. For each of these genes, the threshold cycle (CT) values were measured in each cDNA sample as the cycle number at which the fluorescent signal generated by the cleavage of genespecific fluorogenic TaqMan probe in the course of PCR amplification exceeds a fixed baseline level. The CT for collagens were normalized (⌬CT) by subtracting the CT value for the ARP gene in the same cDNA sample. The use of ARP as a reference gene was validated by the fact that the obtained CT values for ARP were scattered randomly (within ⫾ 0.5 range) among control and FAEE-treated samples, justifying the use of ARP as a housekeeping gene. Next, the ⌬⌬CT values were calculated relative to a control cDNA sample, and the amount of collagen mRNA was determined relative to this control as 2⫺⌬⌬CT (Bulletin #2 for the ABI Prism 7700 Sequence Detection System, under http://www.appliedbiosystems.com/ support/tutorials).36

Gelatin Zymography Gelatinase activity was assessed in both the cell lysate and in the conditioned medium of pancreatic acini by using 10% zymogram gels with 0.1% gelatin (Invitrogen). Aliquots of conditioned media were standardized for total cell protein content. After electrophoresis under nondenaturing conditions, gels were washed for 1 hour in 2.5% Triton X-100 and incubated at 37°C for 16 hours in 50 mmol/L Tris-buffer. Then the gels were stained with 0.5% Coomassie Blue and air-dried. Densitometry analysis of inverted gel pictures was used to analyze relative protease activity (Scion imaging software). Separate control gels were incubated in the assay buffer supplemented with 20 mmol/L ethylenediaminetetraacetic acid or 5 ␮g/mL aprotinin to prove the metalloproteinase or serine protease nature of the gelatinolytic activities, respectively. In some gels, aliquots of recombinant human MMP-2 and MMP-9 (3 ng/lane; Sigma Chemical) were run simultaneously with the samples as positive controls.

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Plasmin and Urokinase Activity Plasmin activity was analyzed as described previously.37 Briefly, aliquots of the cell lysate (400 ␮g total protein) were added to 3 mL of Tris buffer (100 mmol/L Tris, pH 7.5, 10 mmol/L CaCl2). Samples were incubated for 30 minutes at 37°C. Then 80 ␮g of the plasmin substrate D-ValLeu-Lys-AFC.2TA (Enzyme Systems Products, Livermore, CA) were added to the samples, and the formation of 7-amino-4(trifluoromethyl)-coumarin (AFC) by enzyme activity was measured spectrofluorometrically at different time points (␭ex 400 nm; ␭em 505 nm). Plasmin activity was expressed as pmol of AFC generated per mg of total protein per minute. Urokinase (uPA) activity was measured by chromogenic assay using a commercial kit (Chemicon International, Temecula, CA). Briefly, aliquots of the cell lysate were added to 96-well plates containing appropriate amounts of assay buffer and a specific uPA chromogenic substrate (tripeptide with pNA group). After 20 hours of incubation at 37°C, the production of a colored product cleaved from the chromogenic substrate by active uPA was detected at 405 nm. Active uPA was calculated relative to known concentrations of uPA and expressed as activity units/mg of total proteins.

Other Assays Total protein concentration in the acinar samples was measured by Bradford assay (Bio-Rad Laboratories, Richmond, CA). Total DNA was quantified by spectrofluorometric assay38 using Hoeschst-33258 stain and calf thymus DNA as a standard (␭ex 356 nm; ␭em 458 nm). Lactate dehydrogenase activity (LDH) was analyzed using a LDH Detection Kit (Roche, Mannheim, Germany).

Statistical Analysis The values reported represent means ⫾ SEM of the measurements from at least 4 separate acini preparations or cultured PSCs. Immunoblots and zymogram pictures are representative of at least 3 independent experiments. Statistical analysis of the data was performed using the SigmaStat software (SPSS Inc., Chicago, IL). We compared group means by using the unpaired Student t test for pairwise comparison and one-way analysis of variance and Tukey post hoc method for multiple comparisons. A P value ⬍ 0.05 was considered statistically significant.

Results FAEEs Increase Collagen, Laminin, and Fibronectin Levels in Pancreatic Acini To test the effects of FAEEs on ECM protein levels in dispersed pancreatic acini, we used different concentrations (0.5, 1, or 3 mol/L) of an equimolecular mixture of ethyl oleate, ethyl palmitate, and ethyl linoleate. A previous study from our laboratory22 estimated that FAEE concentrations as high as 3 mmol/L were produced in acinar cells treated for 1 hour with 100

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mmol/L ethanol. In the present study, we tested whether these concentrations of FAEEs induced cell death in pancreatic acini after 5 hours of incubation. LDH release to the incubation medium was not altered significantly by 3 mmol/L FAEEs (6% LDH release) compared with control cells (4% LDH release). In addition, we previously showed39 that 3 mmol/L FAEE did not increase but decreased both caspase activation and DNA fragmentation in pancreatic acini. The results suggest that FAEEs, at the concentrations used in our study, did not induce cell death in pancreatic acini, although other possible toxic effects cannot be discarded. We first analyzed total collagen content in rat pancreatic acini treated for 1– 4 hours with FAEEs. Total collagen was quantified in cell homogenates by measuring hydroxyproline concentration normalized to total proteins or total DNA in the samples. Both methods gave similar results, and the data presented here are protein normalized. As shown in Figure 1A, FAEEs at 0.5 mmol/L were ineffective in increasing collagen levels at any of the incubation times tested. However, collagen content significantly increased after 3 or 4 hours incubation with 1 and 3 mmol/L FAEEs as compared with control values. Because 1 and 3 mmol/L FAEEs effectively increased total collagen, we used these concentrations to test the effects of FAEEs on ECM protein content in pancreas. Because FAEEs can be hydrolyzed into free fatty acids in the incubation medium,40 we compared the effect of FAEEs on collagen content using 2 systems of delivery: FAEEs dissolved in DMSO or reconstituted into liposomes. Liposomes allow FAEEs to go directly into the cells and protect them from degradation in the extracellular space. As shown in Figure 1B, both ways of delivery were similarly effective in increasing collagen content in pancreatic acini, suggesting that these ethanol metabolites exert their action by a mechanism inside the cell. We next examined whether free fatty acids, the primary products of FAEE hydrolysis, were responsible for the observed increase in collagen. Thus, we incubated pancreatic acini for 3 hours with a mixture of oleic, linoleic, and palmitic acid, the corresponding fatty acids in our FAEE preparation. As shown in Figure 1C, 1 and 3 mmol/L free fatty acids were ineffective to increase collagen levels in pancreatic acini. We therefore conclude that FAEEs themselves, rather than their degradation products, caused the observed increase in total collagen content in pancreatic acini. Besides collagen, we also analyzed fibronectin and laminin levels in pancreatic acini after 1– 4 hours incubation in the absence or presence of FAEEs. Fibronectin

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decreased with time and at 4 hours were similar between FAEE-treated and control acini. Fibronectin levels significantly increased after treatment with both 1 and 3 mmol/L FAEEs compared with controls. This effect was observed after 2 hours of incubation and, similarly to collagen, persisted in up to 4 hours of incubation (Figure 2B). Maximally effective concentrations of FAEEs (3

Figure 1. Effect of FAEEs and free fatty acids on total collagen content in dispersed pancreatic acini. Total collagen was estimated in cell lysates by measuring hydroxyproline concentration (␮g/mg total protein). Values represent mean ⫾ SEM relative to untreated cells (control). (A) Time-dependent effect of FAEEs on collagen content. Acini were incubated for 1– 4 hours with the indicated concentrations of FAEEs dissolved in DMSO (n ⫽ 4 –7 independent experiments). (B) Acini were treated for 3 hours with FAEEs dissolved in DMSO or incorporated into liposomes (n ⫽ 4 –7). (C) Effect of free fatty acids on total collagen content. Pancreatic acini were treated for 3 hours with an equimolecular mixture of oleic, linoleic, and palmitic acid at the indicated concentrations (n ⫽ 4). *P ⬍ 0.05 vs. control.

and laminin expression were examined by Western blotting using 6% Tris-glycine gels. Relative expression of both ECM proteins was normalized to phospholipase C ␥1 (PLC ␥1) levels. As shown in Figure 2A, both 1 and 3 mmol/L FAEEs markedly increased laminin levels after 2 and 3 hours of incubation However, laminin levels

Figure 2. (A) Laminin and (B) fibronectin protein levels in pancreatic acini. Acini were incubated for 1– 4 hours in the absence and presence of FAEEs at the indicated concentrations. Fibronectin and laminin expression were analyzed in the cell lysates by Western blotting using 6% Tris-glycine gels. The intensity of the bands was quantified by densitometry, normalized to PLC ␥1 levels, and is given relative to controls (mean ⫾ SEM of 3– 6 independent experiments per time point; *P ⬍ 0.05 vs. control). Panels show representative immunoblots for (A) laminin and (B) fibronectin after 3 hours of incubation.

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mmol/L) produced 2.1- and 1.8-fold increases in laminin and fibronectin levels, respectively. Acetaldehyde Increases Fibronectin Levels, But Does Not Alter Collagen and Laminin Content To determine the effects of acetaldehyde on ECM protein content, we measured collagen, laminin, and fibronectin levels in pancreatic acini treated for 1– 4 hours with 0.2 and 1 mmol/L acetaldehyde. As shown in Figure 3, acetaldehyde did not change collagen and laminin levels at any of the incubation times tested. Fibronectin levels significantly increased after 3 hours of incubation with 0.2 and 1 mmol/L acetaldehyde. These results differ from those obtained with FAEEs, showing that the nonoxidative ethanol metabolites effectively increased the levels of all 3 ECM proteins tested. FAEEs Do Not Affect ECM Protein Gene Expression in Pancreatic Acini Having shown that FAEEs increased ECM protein levels in pancreatic acini, the next step was to investigate whether this effect was caused by up-regulation of ECM mRNA expression. Pancreatic acini were treated for 1 and 3 hours with 1 or 3 mmol/L FAEEs, and mRNA expression for collagen ␣1(I), collagen ␣1(III), and laminin ␥2 was measured. We first applied semiquantitative RT-PCR as described previously.35 The results (Figure 4) showed that FAEEs did not cause a significant change in mRNA expression for the ECM proteins measured. We next quantified mRNA expression for the collagens by real-time PCR with the TaqMan method.36 The realtime quantitative PCR data (Table 1) confirmed that FAEEs, under conditions used, did not significantly alter mRNA expression for the ECM proteins studied. These results indicate that FAEEs increase ECM protein content in pancreatic acini by a mechanism different from regulating mRNA levels. FAEEs Increase ECM Protein Levels in Pancreatic Acini by Posttranscriptional Mechanisms To further prove that FAEEs altered ECM protein levels by posttranscriptional mechanisms, we analyzed total collagen and laminin content in pancreatic acini treated with actinomycin D to block gene transcription. Pancreatic acini were preincubated for 1 hour in the presence or absence of 10 ␮g/mL actinomycin D and then incubated for 3 hours more after adding 1 or 3 mmol/L FAEEs. Of note, under these conditions actinomycin D did not induce cell death in pancreatic acini.41

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As shown in Figure 5, actinomycin D failed to affect the FAEE-induced increase in collagen and laminin levels; similar results were obtained for fibronectin (not shown). These data indicate that the stimulatory effects of FAEEs on ECM protein levels are independent of gene transcription. FAEEs, But Not Acetaldehyde, Reduce ECMDegrading Protease Activity The results presented earlier indicate that FAEEs increase ECM protein levels in pancreatic acini without affecting ECM gene transcription. To address whether these increases represent a reduction in ECM degradation, we examined the effect of ethanol metabolites on the major ECM-degrading systems, MMPs, and serine proteases from the Plg system. First, we examined MMP activities in pancreatic acini treated for 1– 4 hours with the ethanol metabolites. MMP activity was analyzed in the conditioned medium by gelatin zymography. Aliquots of recombinant human MMP-2 and MMP-9 were run as positive controls of gelatinase activity. Gels were incubated with aprotinin to block serine protease activities. Figure 6A shows a representative zymogram for 3 hours of incubation. Clear bands appearing in the zymograms corresponded to the presence of gelatinases matching the relative molecular masses of the zymogen proMMP-2 (72 kilodaltons), the active MMP-2 (64 kilodaltons), and another faint band also present in recombinant MMP-2 (⬃58 kilodaltons). Addition of 20 mmol/L ethylenediaminetetraacetic acid to the incubation medium of the gels completely blocked band formation (not shown), indicating that all these bands were MMPs. Interestingly, neither FAEEs nor acetaldehyde affected MMP-2 activity at any incubation time tested. As shown in Figure 6A, MMP-9 activity was not detected in the conditioned medium. We also assessed MMP-2 expression in whole-cell lysate by Western blot analysis. As shown in Figure 6B, treatment with FAEEs or acetaldehyde did not alter the expression of MMP-2 as compared with controls. In conclusion, the results indicate that short-term incubation (1– 4 h) with the ethanol metabolites did not alter MMP-2 activity in pancreatic acini. To investigate further the effects of ethanol metabolites on ECM-degrading proteases, we next examined serine protease activities from the Plg system. Plasmin is an 85-kilodalton serine protease formed by proteolytic activation of plasminogen by specific plasminogen activators such as uPA. Plasmin and uPA participate in tissue remodeling and ECM degradation in physiologic and pathologic conditions.42,43 Therefore, we analyzed

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plasmin and uPA activity in dispersed pancreatic acini treated for 1– 4 hours with FAEEs or acetaldehyde. Plasmin activity was measured in whole-cell lysates by fluorogenic assay (Figure 7A) and gelatin zymography (Figure 7B). As shown in Figure 7A, FAEEs significantly Figure 4. ECM protein mRNA expression in pancreatic acini in response to FAEEs. Pancreatic acini were incubated for 1 or 3 hours without and with FAEEs at indicated concentrations. Total RNA was isolated and mRNA expression of collagen ␣1(I), collagen ␣1(III), laminin ␥2 , and the housekeeping ARP gene was measured with semiquantitative RT-PCR. The results are representative of 3 similar experiments performed on different preparations of acini.

reduced plasmin activity by 35% after 1 hour of incubation, and the inhibitory effect persisted for up to 3 hours. Figure 7B shows a representative gelatin zymogram after 3 hours of incubation with the ethanol metabolites. Zymogram gels were incubated in the absence or presence of the serine protease aprotinin (5 ␮g/mL). Two prominent bands of serine protease activity can be visualized in the zymograms: one at approximately 100 kilodaltons and the second at a position near 85 kilodaltons. FAEEs markedly reduced the activity of the 85kilodalton protease, likely plasmin. Complete inhibition of these protease activities by aprotinin revealed the presence of faint bands corresponding to MMP activity (Figure 7B, right panel). In contrast to the results obtained with FAEEs, acetaldehyde did not affect plasmin activity at any of the incubation times tested (Figure 7). To further prove that the increases in ECM protein levels induced by FAEEs were caused by serine protease inhibition, pancreatic acini were treated for 3 hours with FAEEs in the absence or presence of aprotinin. Laminin and fibronectin levels were measured by Western blot analysis. In the absence of aprotinin, FAEEs increased laminin and fibronectin levels as compared with controls (Figure 7C). Aprotinin treatment by itself increased basal levels of both ECM proteins and prevented any stimulatory effect of FAEEs. The results suggest that FAEEs and aprotinin increased ECM protein levels by Š Figure 3. Effect of acetaldehyde on ECM protein levels in pancreatic acini. Acini were treated for 1– 4 hours in the absence and presence of acetaldehyde (AA) at indicated concentrations. (A) Total collagen content was determined by hydroxyproline measurement and is expressed relative to controls (mean ⫾ SEM of 5–7 independent cell preparations). (B) Laminin and (C) fibronectin expression were determined by Western blotting. The intensity of the ECM protein bands in the immunoblots was quantified by densitometry, normalized to PLC ␥1 levels, and is given relative to controls (mean ⫾ SEM of 3– 6 independent experiments per time point: *P ⬍ 0.05 vs. control). (B and C) Representative immunoblots for 3 hours of incubation.

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Table 1. Collagen mRNA Expression in Dispersed Pancreatic Acini Collagen ␣1 (III)

Collagen ␣1 (I) Time point 1h

3h

Treatment Control 1 mmol/L 3 mmol/L Control 1 mmol/L 3 mmol/L

FAEE FAEE FAEE FAEE

⌬CT

⌬⌬CT

mRNA relative amount

⌬CT

⌬⌬CT

mRNA relative amount

5.00 ⫾ 0.16 4.66 ⫾ 0.24 4.82 ⫾ 0.34 4.81 ⫾ 0.14 4.78 ⫾ 0.49 4.65 ⫾ 0.35

0 ⫾ 0.22 ⫺0.34 ⫾ 0.29 ⫺0.18 ⫾ 0.37 ⫺0.19 ⫾ 0.21 ⫺0.22 ⫾ 0.51 ⫺0.35 ⫾ 0.37

1.0 1.27 1.13 1.14 1.16 1.27

6.37 ⫾ 0.41 6.41 ⫾ 0.29 6.22 ⫾ 0.47 6.56 ⫾ 0.24 6.20 ⫾ 0.41 6.42 ⫾ 0.36

0 ⫾ 0.57 0.04 ⫾ 0.50 ⫺0.15 ⫾ 0.62 0.19 ⫾ 0.47 ⫺0.17 ⫾ 0.57 0.05 ⫾ 0.54

1.0 0.97 1.11 0.88 1.13 0.97

NOTE. Real-time RT-PCR was performed using TaqMan assay as described in the Materials and Methods section. The comparative CT (threshold cycle) method was used for relative mRNA quantification. ⌬CT represents the difference in CT values for collagen gene and the reference ARP housekeeping gene in the same cDNA sample. The amount of collagen mRNA was calculated relative to the 1-hour control (calibrator) as 2⫺⌬⌬CT Values are means ⫾ SD from triplicate measurements. Similar results were obtained on another preparation of acini.

the same mechanism, likely a reduction in serine protease activity. Because the serine protease uPA is one of the main physiologic plasminogen activators, we examined whether the observed inhibitory effect on plasmin activity is caused by a reduction in uPA expression or activity. uPA expression was analyzed by Western blotting using

Figure 5. (A) Collagen and (B) laminin levels in pancreatic acini after blocking gene transcription with 10 ␮g/mL actinomycin D. Pancreatic acini were treated for 60 minutes with and without 10 ␮g/mL actinomycin D and then incubated for an additional 3 hours with FAEEs. Total collagen content was estimated by hydroxyproline content and laminin levels were analyzed by Western blotting. PLC ␥1 expression was used to confirm equal protein loading in the immunoblots. Results are representative of 1– 4 independent experiments (*P ⬍ 0.05 vs. control).

an antibody that recognizes 2 forms of the enzyme: the single-chain proenzyme form (54 kilodaltons) and the 2-chain active form (heavy chain, 35 kilodaltons; and light chain, 19 kilodaltons). Pancreatic acini express both forms of the enzyme as well as the uPA receptor (Figure 8A). As shown in Figure 8B, FAEEs did not alter the expression of uPA or its receptor but significantly inhibited uPA activity to the same extent as they inhibited plasmin activity (see Figure 7B). Similar to the results obtained for plasmin, the inhibitory effect of FAEEs on uPA activity was attenuated at 4 hours of incubation.

Figure 6. MMP activity in pancreatic acini treated for 1– 4 hours with ethanol metabolites. (A) The conditioned media were analyzed by gelatin zymography. Human recombinant MMP-2 and MMP-9 aliquots were run in the gels as positive controls. Shown is a representative zymogram showing MMP-2 activity in the conditioned medium after 3 hours of incubation with FAEEs or acetaldehyde at indicated concentrations. Similar zymograms were obtained for all incubation times tested. (B) Western blot analysis of MMP-2 in cell lysates from pancreatic acini. A representative immunoblot of active MMP-2 after 3 hours of incubation with the ethanol metabolites. Tubulin expression was used to confirm equal protein loading. Panels are representative of 4 independent experiments.

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Figure 7. Serine protease activity in pancreatic acini treated for 1– 4 hours with 1 and 3 mmol/L FAEE or 0.2 mmol/L acetaldehyde (AA). (A) Plasmin activity was measured in cell lysates using a specific fluorogenic substrate as described in the Materials and Methods section (n ⫽ 3– 4 per group and time point, *P ⬍ 0.05 vs. control). (B) Representative gelatin zymogram showing serine protease activity in cell lysates after 3 hours of incubation. Gels were incubated in the absence or presence of the serine protease inhibitor, aprotinin. Faint bands corresponding to MMP activity were visualized in the right panel after complete inhibition with aprotinin. (C) Western blot analysis of laminin and fibronectin in pancreatic acini treated with FAEEs in the absence or presence of aprotinin. PLC ␥1 expression was used to confirm equal protein loading. (B and C) Representative of 5 independent experiments.

Taking all the results together, we conclude that FAEEs induced changes in ECM protein levels in pancreatic acini likely by inhibition of ECM serine protease(s) such as plasmin, and without affecting key MMPs. FAEEs Increase ECM Protein Levels and Decrease Plasmin and uPA Activities in Rat Pancreas To address whether FAEEs affect ECM protein content and ECM-degrading proteases in pancreas in vivo, we treated anesthetized rats for 6 hours with a continuous intravenous infusion of 0.3 mmol/kg/h FAEEs reconstituted into liposomes. Control animals were treated with empty liposomes. At the end of the perfusion period, pancreas was removed and analyzed for ECM protein content and plasmin and uPA activities. Histologic analysis of H&E-stained pancreatic sections showed that FAEEs did not cause noticeable tissue damage (not shown). As shown in Figure 9A, FAEEs increased laminin and fibronectin levels by 1.5- and 1.4fold, respectively. Similar to what we observed in pancreatic acini (Figures 7 and 8), FAEEs significantly decreased plasmin and uPA activities in pancreas homogenates (Figure 9B and C). MMP-2 activity, as measured by gelatin zymography, was not affected by the ethanol metabolites (not shown).

Figure 8. Urokinase expression and activity in pancreatic acini treated for 1– 4 hours with 1 and 3 mmol/L FAEE. (A) Representative immunoblot of uPA and uPA receptor (UPAR) expression in pancreatic acini treated with FAEEs for 3 hours. uPA antibody recognizes the proenzyme (1 chain) and the active form (2-chain protein) of the enzyme. Shown are representative immunoblots from 3 independent experiments. (B) uPA activity was measured in cell lysates using a specific chromogenic assay (n ⫽ 3–5 per time point, *P ⬍ 0.05 vs. control).

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Figure 9. Effect of intravenous administration of 0.3 mmol/kg/h FAEEs on ECM turnover in pancreas. (A) Laminin and fibronectin expression was analyzed by Western blotting. PLC ␥1 expression was used to confirm equal protein loading. Each lane represents an individual animal. (B) Plasmin and (C) uPA activities were measured in pancreas homogenates using specific substrates (n ⫽ 6, *P ⬍ 0.05 vs. control).

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sponsible for the increase in ECM proteins found in the acinar preparations treated with ethanol metabolites. To further investigate the role of PSCs in the observed effects of FAEEs, we studied changes in ECM protein levels in cultured PSCs (passage 1– 4) treated for 3 hours with the ethanol metabolites. As shown in Figure 11, neither FAEEs nor acetaldehyde increased laminin levels in PSCs at any of the passages tested (Figure 11A and B). Both ethanol metabolites decreased collagen synthesis in PSCs compared with controls (Figure 11C). FAEEs did not affect fibronectin levels at any of the passages tested (Figure 11D). Acetaldehyde increased fibronectin levels, but only after passage 4. Furthermore, 3 hours of incubation with the ethanol metabolites did not affect the levels of ␣-SMA in cultured PSCs. Taken together, these results show that short-term incubation with the ethanol metabolites did not increase ECM protein levels in PSCs, suggesting that acinar cells rather than PSCs are responsible for the observed effects on ECM protein levels found in pancreatic acini.

Discussion Short-Term Incubation Ethanol Metabolites Does Not Increase ECM Protein Levels in Cultured PSCs It has been reported that activated PSCs can synthesize large amounts of ECM proteins14,16,44 and also express components of ECM degrading systems.10,17 Therefore, we investigated whether PSCs present in acinar preparations could be responsible for the observed increases in ECM protein levels induced by FAEEs in pancreatic acini. To determine whether activated PSCs were present in our preparations of pancreatic acini, we analyzed immunoreactivity to desmin and ␣-SMA in pancreatic acini over the course of several days in culture as described in the Materials and Methods section. As shown in Figure 10A, 3 hours after seeding, acini were desmin and ␣-SMA negative. By 12 hours after seeding, some desmin-positive spindle-shaped cells had migrated from the acini and begun to proliferate (Figure 10B). During the next 2–5 days in culture, acinar cells gradually disappeared (Figure 10C), and eventually were replaced completely by desmin-positive cells with a myofibroblastic phenotype (Figure 10D). Passaged cells remained desmin-positive (Figure 10E), and all were ␣-SMA positive (Figure 10F ). These data indicate that the PSCs present in freshly isolated pancreatic acini exhibit a resting phenotype that persisted throughout the shortterm incubations used in this study. Therefore, we conclude that these quiescent PSCs are unlikely to be re-

It generally is accepted that fibrosis results from a failure in repair mechanisms after organ injury.45 A coordinated biosynthesis and proteolytic clearance of matrix components are central to organ repair. Alterations in this coordinated process lead to an abnormal increase

Figure 10. Immunohistochemistry analysis of desmin (A–E) and (F ) ␣-SMA expression in pancreatic acini and PSCs. PSCs were outgrown from dispersed rat pancreatic acini as described in the Materials and Methods section. Figures show desmin expression in pancreatic acini (A) 3 hours, (B) 24 hours, (C) 2 days, and (D) 4 days after seeding. After 2–5 days in primary culture, acinar cells were replaced totally by desmin-positive cells with a myofibroblastic phenotype (original magnification, 400⫻). (E and F ) Desmin (after passage 1) and ␣-SMA (after passage 3) expression, respectively (original magnification 100⫻).

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Figure 11. Effect of ethanol metabolites on ECM protein levels in cultured PSCs. Western blot analysis for laminin and ␣-SMA in PSCs (passages 1 and 3) incubated for 3 hours in the absence or presence of (A) FAEEs or (B) acetaldehyde at the indicated concentrations. Also shown are laminin and ␣-SMA expression in untreated pancreatic acini preparations (AC). (C) Collagen synthesis in PSCs assessed by measuring 14C-proline incorporation into collagenase-sensitive proteins. Results are expressed as percent of controls (n ⫽ 4; *P ⬍ 0.05 vs. control). (D) Western blot analysis for fibronectin in PSCs (passage 4) treated for 3 hours with FAEEs or acetaldehyde at the indicated concentrations. Immunoblots are representative of 3 independent experiments.

in ECM protein synthesis and/or a decrease in ECM degradation, and, ultimately, to fibrosis.46,47 Although there has been a remarkable progress in the understanding of the fibrotic processes, the mechanisms underlying ethanol-induced fibrosis in pancreas remain unclear. Ethanol metabolites have been proposed as mediators of ethanol damage. In normal pancreas, ethanol is comparably metabolized to acetaldehyde and fatty acid ethyl esters.22 Although acetaldehyde has been shown to be a potential factor in the induction of fibrogenesis,15,21,28,48 –50 the role of FAEEs in the fibrotic process is unknown. In the present study, we investigated the fibrotic response to FAEEs and acetaldehyde in rat pancreatic acini as well as in pancreas in vivo. We used ethyl palmitate, ethyl oleate, and ethyl linoleate because they represent the predominant FAEEs found in human blood after ethanol ingestion.51 The concentrations of FAEEs used in this study are based on estimates of FAEE intracellular concentrations found in rat acini after 1 hour of incubation with 100 mmol/L ethanol.22 Acetaldehyde was used at concentrations similar to those reported in rats after ethanol administration.52 Our studies show that FAEEs have fibrogenic effects in rat exocrine pancreas. FAEEs significantly increased protein levels of collagen, laminin, and fibronectin both in

pancreatic acini and in pancreas in vivo. This increase was not caused by up-regulation in gene expression for the ECM proteins or activation of PSCs present in the acinar preparation, but rather by an inhibitory effect of FAEEs on ECM degradation. FAEEs significantly inhibited the activity of the serine protease, plasmin, and that of the plasminogen activator, urokinase (uPA). Furthermore, intravenous administration of FAEEs to rats also increased ECM protein levels and reduced the activities of uPA and plasmin in pancreas. Taken together, the results suggest that FAEEs increase ECM protein levels through inhibition of serine protease activities of the Plg system. FAEEs increased ECM protein levels in cultured acini within 2 hours incubation and the effect persisted up to 4 hours. Similarly, FAEEs significantly reduced the activities of ECM-degrading proteases within 1 hour of incubation and the effect was attenuated after 4 hours. It has been reported that FAEEs are hydrolyzed rapidly to free fatty acids.40,53 As shown in Figure 1C, free fatty acids had no effect on ECM protein content. These results therefore suggest that progressive degradation of FAEE explains the lack of a longer lasting effect in response to a single exposure. However, the observed fibrogenic effects of FAEEs may be relevant in ethanolinduced pancreatic fibrosis in humans because FAEE can

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be produced constantly during long-term ethanol consumption.24 The importance of FAEEs for the development of fibrosis within the injured pancreas will depend on their rate of formation and the duration of ethanol exposure. Although FAEEs alter ECM protein levels, our findings indicated that they do not significantly affect mRNA expression for ECM proteins under study. Although minor changes in the mRNA expression cannot be excluded, the results—in particular, the quantitative real-time PCR data—indicate that the FAEE-induced increase in ECM protein content we have observed cannot be accounted for by transcriptional up-regulation. Furthermore, blocking gene transcription with actinomycin D did not abolish the increase in ECM induced by FAEEs. These results suggest that a reduction in ECM degradation is a likely mechanism to explain the observed effects of FAEEs on ECM protein levels in pancreas. We examined the effect of ethanol metabolites on 2 major ECM-degrading systems: serine proteases from the Plg system and MMPs, in particular MMP-2 and MMP-9. The results indicate that although MMPs were not affected, FAEEs significantly inhibited serine proteases from the Plg system. This system comprises the proenzyme plasminogen that is activated by specific activators to plasmin. Plasmin is a broad-specificity serine protease, which degrades several ECM proteins and is involved in the activation of MMPs and growth factors such as the profibrotic cytokine transforming growth factor ␤1.42 The serine protease uPA is considered the main Plg activator in the extravascular space. The binding of the inactive pro-uPA to the specific uPA receptor in the plasma membrane causes the activation of the enzyme and localizes the proteolytic activity of uPA and plasmin on the cell surface.54 The Plg system is associated with matrix remodeling in physiologic and pathologic conditions.7,8,55–59 In pancreas, Plg activation has been suggested to play an important role in both acute60 and chronic pancreatitis.9 Of note, pancreatic tissue from patients with chronic alcohol-induced pancreatitis has an increased fibrogenic activity and an impaired ECM-degradation capacity.4 Interestingly, alcohol consumption in humans is associated with changes in the Plg system.61– 65 Thus, the Plg system appears to be an important target to investigate the mechanisms implicated in ethanol-induced fibrosis. Our results showed that FAEEs inhibited uPA activity both in isolated pancreatic acini and in pancreas in vivo. As expected, the inhibition of uPA activity led to a significant decrease in plasmin activity and this effect

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correlated with a concomitant increase in ECM protein levels. The precise mechanism underlying the effects of FAEEs on the Plg system in pancreas remains to be elucidated. Control levels of plasmin activity were similar in the presence or absence of FAEEs directly added to the assay (data not shown), suggesting that FAEEs do not affect the enzyme directly. Although there are no reports on the effects on FAEEs on ECM turnover, a variety of toxic cellular effects have been attributed to FAEEs, including decreased DNA and protein synthesis,66 mitochondrial dysfunction affecting the energy status,26 lysosomal fragility,30 and altered cell membrane functions.67 Werner et al.31 reported that FAEE administration to rats produced pancreatic damage marked by transient edema, trypsinogen activation, and vacuolization of the cytoplasm. A recent study from this group68 showed that ethanol infusion to rats in the presence of inhibitors of oxidative metabolism increased plasma and tissue levels of FAEEs that correlated with pancreatic injury. These reports showed that FAEEs are involved in pancreatic cell damage induced by alcohol. The results of our study suggest that FAEEs play an important role in pancreatic fibrosis by inhibiting ECM degradation. In contrast to the results obtained for FAEEs, shortterm incubation with acetaldehyde had minor effects on ECM production in pancreatic acini. These results differ from those reported previously for acetaldehyde in other cell types.15,28,48,49 The differences may be owing to the fact that our study examined the effect of short-term incubation with acetaldehyde, whereas most of the studies showing an increase in ECM expression with acetaldehyde were performed on myofibroblast-type cells after 24 or 48 hours of incubation. Moshage et al.49 reported that acetaldehyde significantly increased type I collagen production in liver fat-storing cells but not in cultured hepatocytes, supporting the notion that acetaldehyde effects on ECM may be cell specific. The present study mainly addressed the fibrogenic effects of ethanol metabolites on pancreatic acini. Acinar cells are the major cell type in pancreatic acini but a small number of quiescent PSCs also are present in acinar preparations. Because active PSCs represent the major source of ECM in pancreatic fibrosis12,16,17,44 and express components of ECM-degrading systems,10,17 these cells could have been responsible for the increase in ECM content found in pancreatic acini in response to FAEEs. To investigate this issue, we cultured PSCs outgrown from our acinar preparations and treated them with the ethanol metabolites in the same conditions as those for pancreatic acini. Unlike the results obtained with pancreatic acini, we found that short-term incubation (3 h)

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with FAEEs or acetaldehyde did not increase ECM protein levels in PSCs. It has been reported previously that acetaldehyde stimulated collagen synthesis in rat PSCs but after 24 and 48 hours of incubation.15 Therefore, our results suggest that acinar cells rather than PSCs are responsible for the increases in ECM protein levels induced by short-term incubation with FAEEs in dispersed pancreatic acini. In conclusion, the present results suggest that the nonoxidative ethanol metabolites, FAEEs, play a significant role in ethanol-induced pancreatic fibrosis. These data reinforce the importance of FAEEs produced with alcohol consumption in tissue repair processes within the injured pancreas. It has been suggested that ethanoldamaged acinar cells participate in the fibrotic process by releasing cytokines that in turn activate PSCs, leading to an increase in ECM protein production.15 However, other mechanisms also play an important role in the development of fibrosis. Thus, the results of the present study suggest that ethanol metabolites can directly modulate ECM turnover and degradation through inhibition of ECM-degrading proteases from acinar cells. Persistent alterations in ECM metabolism and activation of PSCs induced by long-term alcohol consumption may lead to pancreatic fibrosis.

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13. 14.

15.

16.

17.

18.

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sata M. Linkage of oxidative and nonoxidative ethanol metabolism in the pancreas and toxicity of nonoxidative ethanol metabolites for pancreatic acinar cells. Surgery 2001;129:736 –744.

Received August 28, 2002. Accepted August 28, 2003. Address requests for reprints to: Aurelia Lugea, Ph.D., Veterans

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Affairs Greater Los Angeles Healthcare System, Building 258, Room 339, 11301 Wilshire Boulevard, Los Angeles, California 90073. e-mail: [email protected]; fax: (310) 268-4578. Supported by the Department of Veterans Affairs and the Research Center for Alcoholic Liver and Pancreatic Diseases (P50AA11999), and funded by the National Institute on Alcohol Abuse and Alcoholism.