Nonoxidative ethanol metabolites alter extracellular matrix protein content in rat pancreas  Aurelia Lugea, Ilya Gukovsky, Anna S Gukovskaya, Stephen J Pandol  Gastroenterology  Volume 125, Issue 6, Pages 1845-1859 (December 2003) DOI: 10.1053/j.gastro.2003.09.021

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 (μg/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. Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

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 γ1 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. Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

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 γ1 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. Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

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 α1(I), collagen α1(III), laminin γ2, 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. Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

Figure 5 (A) Collagen and (B) laminin levels in pancreatic acini after blocking gene transcription with 10 μg/mL actinomycin D. Pancreatic acini were treated for 60 minutes with and without 10 μg/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 γ1 expression was used to confirm equal protein loading in the immunoblots. Results are representative of 1–4 independent experiments (∗P < 0.05 vs. control). Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

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. Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

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 γ1 expression was used to confirm equal protein loading. (B and C) Representative of 5 independent experiments. Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

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). Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

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 γ1 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). Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

Figure 10 Immunohistochemistry analysis of desmin (A-E) and (F) α-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 α-SMA (after passage 3) expression, respectively (original magnification 100×). Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)

Figure 11 Effect of ethanol metabolites on ECM protein levels in cultured PSCs. Western blot analysis for laminin and α-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 α-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. Gastroenterology 2003 125, 1845-1859DOI: (10.1053/j.gastro.2003.09.021)