Vitamin E attenuates biochemical and morphological features associated with development of chronic pancreatitis

José-Antonio Gómez,1 Xavier Molero,1 Eva Vaquero,1 Ana Alonso,1 Antonio Salas,2 and Juan-R. Malagelada1

1Digestive System Research Unit, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, 08035 Barcelona; and 2Hospital Mutua, 08221 Terrassa, Spain

Submitted 4 August 2003 ; accepted in final form 1 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
The objective was to investigate the effects of vitamin E on collagen deposition induced by Cyclosporin A (CsA) administration in rats with caerulein (Cr) pancreatitis. CsA transforms the fully regenerative, self-limited form of Cr pancreatitis into a chroniclike disease in conjunction with increased transforming growth factor (TGF)-{beta} and myofibroblast proliferation. Vitamin E inhibits TGF-{beta} release in mesangial cells and reduces CsA cytotoxicity. Wistar rats received CsA daily (20 mg/kg), and CR pancreatitis was induced on days 1 and 8 (Cr + CsA group). In a separate group, vitamin E (600 mg·kg–1·day–1) was administered starting 4 days before CsA. Three other groups received either vehicle, CsA, or Cr alone. Thiobarbituric acid-reactive substance (TBARS), 8-isoprostanes, and hyaluronic acid were measured in plasma obtained on the day the animals were killed (day 15). Pancreases were weighed and processed for light microscopy to assess connective tissue and myofibroblast number. Pancreatic homogenates were also assayed for collagen (hydroxyproline) and TBARS content. TBARS, 8-isoprostane, and TGF-{beta} were elevated in CsA and Cr + CsA rats. Vitamin E treatment greatly decreased these parameters. Vitamin E also decreased the fall in pancreatic weight observed in Cr + CsA pancreas. Pancreatic hydroxyproline and plasma hyaluronic acid were increased in Cr + CsA rats but were effectively reduced by vitamin E. Morphology showed improvement in fibrosis score and a decreased number of myofibroblasts in vitamin E-treated rats. Vitamin E reduces oxidative stress and collagen deposition during the development of experimental chronic pancreatitis. Adjuvant antioxidants may be of value in the treatment of chronic pancreatitis.

cyclosporin; caerulein; myofibroblasts; fibrogenesis


CAERULEIN (Cr) hyperstimulation induces a self-limited form of acute pancreatitis in rats with transient acinar atrophy, limited rise in transforming growth factor (TGF-{beta}), and fibroblast proliferation (13, 44), which are invariably followed by complete restoration of pancreatic integrity. Simultaneous administration of cyclosporin A (CsA) impairs pancreatic repair and results in a chroniclike disease featuring extended acinar atrophy, persistent TGF-{beta} release, increased collagen deposition and collagenase activity, and excess number of activated myofibroblasts (53).

CsA propensity to induce fibrosis formation is well recognized (24). Newly formed fibrosis often leads to early failure of a transplanted organ.

CsA nephrotoxicity is thought to be due to arteriolar vasoconstriction, tubule cell atrophy, and interstitial fibrosis (28, 50). CsA stimulates collagen synthesis in renal fibroblasts and promotes PDGF and TGF-{beta} secretion by tubule cells, two cytokines involved in fibroblast activation (25). On the contrary, CsA exhibits antiproliferative and proapoptotic features in cells other than fibroblasts, such as tubulointerstitial, epithelial, and endothelial cells (14, 50).

The precise molecular mechanisms responsible for CsA-induced fibrogenesis are still undefined. They may involve impaired dephosphorylation of activated key proteins (27, 45), mitochondrial membrane permeability transition pore dysfunction (10), or enhancement of oxidative stress (29, 42, 56).

Vitamin E ({alpha}-tocopherol) is a lipophilic endogenous molecule with important antioxidant and nonantioxidant functions. Working in close connection with ubiquinone, vitamin C, and thiol redox cycles (such as glutathione and lipoic acid), vitamin E plays a leading role in controlling excess oxidative radical formation at cell membranes (9, 40), including mitochondrial membranes (8, 31, 59). Other physiological functions of vitamin E relevant to our study, which are partly or totally independent of its antioxidant capacity, include restoration of TGF-{beta}- and Fas-dependent apoptosis signaling in cancer cells (23, 58), inhibition of superoxide generation in neutrophils (26), decrease in collagenase expression in fibroblasts (43), and inhibition of stimulus-dependent increases in PKC activity, TGF-{beta} bioactivity, and collagen and fibronectin synthesis in mesangial cells (49).

Several studies (2, 42, 56) have demonstrated beneficial effects of vitamin E on CsA cytotoxicity, and recent research suggests that vitamin E may prevent ethanol and acetaldehyde activation of pancreatic stellate cells in culture (4) and ethanol-induced mitochondrial DNA degradation in the liver (34). On the basis of the above-mentioned evidence, we reasoned that vitamin E might prevent or mitigate the adverse effects of CsA on pancreatic regeneration and collagen deposition after Cr pancreatitis.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
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Materials

Cr, hydroxyproline, malonaldehyde bis(dimethylacetal), vitamin E (RRR-{alpha}-tocopherol), and anti-{alpha}-smooth muscle actin monoclonal antibody were purchased from Sigma (St. Louis, MO). BCA reagent and bovine albumin standard for protein determination were from Pierce (Rockford, IL). Labeled streptavidin-biotin (LSAB) kit was from DAKO (Carpinteria, CA). CsA was from Sandoz Pharma (Barcelona, Spain). TGF-{beta}1 test kit was from Genzyme (Cambridge, MA). 8-Isoprostane enzyme immunoassay (EIA) Kit and 8-Isoprostane affinity column were from Cayman Chemical (Ann Arbor, MI). Total lipids colorimetric test kit was from Spinreact (Sant Esteve de Bas, Spain).

Hyaluronic acid was measured with the Pharmacia HA Test radiometric assay kit (Pharmacia and Upjohn Diagnostics, Uppsala, Sweden). All other chemicals were reagent grade and were obtained from common commercial suppliers.

Methods

Animals and experimental design. All experiments were performed in 300-g male Wistar rats housed in rack-mounted cages under conditions of constant temperature and a 12:12-h light-dark cycle. All procedures were approved by the Animal Experimentation Committee of the Institut de Recerca Vall d'Hebron.

A previously reported method (53) was used for induction of chroniclike pancreatic disease. Briefly, daily intraperitoneal doses of CsA (20 mg/kg diluted 1:4 in saline) were administered for 15 days, starting at day 0. On days 1 and 8, acute pancreatitis was induced by four intraperitoneal injections of Cr (20 µg/kg) at 1-h intervals (Cr + CsA group). Rats were euthanized at day 15, and pancreases were excised for histopathological examination (n = 10) or biochemical assays (n = 10). Three other groups underwent the same experimental procedure, except that they received either CsA, Cr, or vehicle (control group) alone. To examine the effects of vitamin E on pancreatic repair after acute pancreatitis, a separate group of rats followed the same protocol as the Cr + CsA group except that a high dose of vitamin E was administered daily by gavage (600 mg/kg), starting four days before the first pancreatitis induction until completion of the experiment (Cr + CsA + VitE group). To compensate for the stress associated with animal manipulation, all other groups received an equivalent volume of saline by gavage on a daily basis. Dietary vitamin E has been shown to increase vitamin E tissue concentration in a dose-dependent manner (19). Better results are often obtained when high doses are administered (6, 21, 51), whereas adverse side effects only occur with extremely high doses for long periods of time (1). Before rats were euthanized, blood was withdrawn by cardiac puncture using heparinized needles and syringes and placed in siliconized tubes, and plasma was obtained after low-speed centrifugation of total blood. Special care was taken in the process of blood manipulation, from extraction to plasma separation, to minimize the risk of cell lysis. Samples obtained to measure {alpha}-tocopherol, thiobarbituric acid-reactive substance (TBARS), or 8-isoprostanes were protected from oxidation by adding butylated hydroxytoluene (0.01%) during the extraction. Plasma samples were freed from platelets by means of an additional centrifugation (5,800 g for 10 min at 4°C) to avoid platelet interference with the TGF-{beta}1 assay.

To verify that vitamin E treatment did not reduce acute pancreatitis severity, Cr + CsA rats and Cr + CsA + VitE rats (n = 5/group) were euthanized 6 h after acute pancreatitis induction. Amylasemia, tissue edema, and myeloperoxidase activity were measured.

Pancreatic edema was assessed by measuring pancreatic water content as the percentage of total wet weight. A sample of the head of the pancreas was wet weighed, desiccated at 160°C for 24 h, and reweighed.

Biochemical determinations. Amylase activity was assessed by the enzymatic colorimetric determination of pancreatic {alpha}-amylase (Sentinel, Milan, Italy) using an automated analyzer (Olympus AU5400; Olympus Optical, Tokyo, Japan). Aliquots (200 µl) of 1:50 dilution of the sample were used in the assay.

Tissue myeloperoxidase activity was measured in pancreatic homogenates using a previously-described method (54).

Plasma D-{alpha}-tocopherol was determined by HPLC. Briefly, 200 µl of plasma were placed in acid-washed test tubes. After deproteinization with ethanol (400 µl), 2 ml hexane were added and the mixture was vortexed for 60 s and then centrifuged for 20 min at 4°C. Supernatants were dried under nitrogen, resuspended in 200 µl methanol, and subjected to reverse-phase HPLC equipped with a 440 diode array detector (Kontron Instruments, Zurich, Switzerland). Detection of {alpha}-tocopherol was accomplished with ultraviolet detection at 284 nm. Calibration of the HPLC system was performed using fresh solutions of {alpha}-tocopherol in ethanol.

For hydroxyproline assay, total pancreases were excised, trimmed of fat and lymph nodes, weighed, frozen, and lyophilized. A powder was made in a mortar from the dried residue and resuspended in ice-cold distilled water (100 mg/ml). Pancreatic homogenates were prepared by serial use of a motor-driven shearer (Tissue Tearor 985–370, Biospec Products, Drewel, WI) and Dounce tissue homogenizers on ice.

Total protein and hydroxyproline content were quantified from crude homogenates. Protein concentration was determined by bicinchoninic acid reagent (Pierce).

Hydroxyproline content was determined as described (50). Briefly, 200 µl of crude homogenate were placed in an ampoule containing 2 ml 6 N HCl and sealed. Hydrolysis was performed at 110°C for 16 h. The hydrolysate was dried, and the sediment was redissolved in 2 ml distilled water and dried again. This latter step was repeated three times before the residue was resuspended in 3 ml 0.2 M citrate buffer (pH 2.2). One milliliter of the sample or of hydroxyproline standards was placed in a pyrex test tube and mixed with 1 ml chloramine T solution prepared as described by Rojkind and Gonzalez (45). After 20 min, the reaction was stopped by the sequential addition of 0.5 ml sodium thiosulphate (2 M), 1 ml NaOH (1 N), and 2 g NaCl. Proline was extracted in 2.5 ml toluene by shaking the contents for 30 s. The aqueous fraction was placed in a boiling water bath for 30 min which was then cooled to room temperature. Hydroxyproline was extracted in 2.5 ml toluene and then determined by the use of Ehrlich's reagent.

To measure biologically active TGF-{beta}1 concentration in platelet-free plasma, samples and standards were acidified using HCl for 1 h and then neutralized to pH 7.0–7.4 immediately before sample loading for enzyme-linked immunosorbent assay (Genzyme, Cambridge, MA) (30).

The soluble nonprotein component of the extracellular matrix (ECM) hyaluronic acid has been found to be a useful marker of fibrosis (16, 33, 39, 48). Hyaluronic acid was measured in plasma as a collagen-independent marker of increased ECM turnover by means of a radiometric assay (Pharmacia HA test).

Plasma lipid peroxidation was assessed by measuring both TBARS(17) and 8-epi PGF2{alpha} (8-isoprostanes), an eicosanoid of nonenzymatic origin produced by direct oxidation of phospholipids by reactive oxygen species (ROS) (32).

Thiobarbituric-reactive substances were measured in platelet-free plasma following published procedures (47, 60). Briefly, 100 µl of samples were added to a reaction mixture containing 700 µl H2O, 200 µl SDS 8.1%, 1,500 µl of 20% acetic acid (pH 3.5), and 1,500 µl of 0.8% thiobarbituric acid. Samples were then heated to 95°C for 1 h and centrifuged at 3,000 g for 15 min. Absorbance of the clear supernatants was determined spectrophotometrically at 532 nm. Standard curves were prepared using malonaldehyde bis(dimethylacetal).

TBARS were also determined in pancreas. Briefly, fresh pancreatic tissue was minced and homogenized (100 mg/ml) in ice-cold 1.15% KCl containing 0.01% butylated hydroxytoluene and stored at –80°C until assayed. Homogenates (100 µl) were assayed in duplicate for TBARS content. From the initial homogenate, 200 µl were dessicated to dryness by heating at 160°C for 16 h and then weighed. Results are provided as nanomoles per 100 mg dry wt.

Total plasma 8-epi PGF2{alpha} was determined using a commercially available EIA kit (Cayman Chemical) according to the manufacturer's specifications. Samples were purified with the help of affinity columns (Cayman) before the EIA assay.

Morphology

Pancreatic specimens were fixed in 10% formalin and embedded in paraffin. Several sections were cut and stained with hematoxylin and eosin (H & E). Connective tissue accumulation was assessed using a semiquantitative morphological scoring system adapted from Demols et al. (12) that grades 0 for normal pancreas and 3 for extensive interstitial fibrosis. Histological gradings were performed on 10 successive fields within each section at a magnification of x100. The mean of the scores of all fields was recorded for each specimen. Acinar atrophy was also scored accordingly (12), taking 0 for normal pancreas and 3 for extensive acinar atrophy. When indicated, immunohistochemistry was performed with the monoclonal anti-{alpha}-smooth muscle actin ({alpha}SMA) antibody to identify myofibroblasts, i.e: interstitial nonvascular {alpha}SMA-positive cells (SMAc) with fibroblastlike shape, using the avidin-biotin-peroxidase complex system (LSAB kit; DAKO). Quantitative analysis of interstitial SMAc was performed at x400 final magnification. Ten nonoverlapping fields were evaluated for each animal in each treatment group.

All histological slides were examined by the participating pathologist (A. Salas), who was unaware of the tissue source.

Statistical Analysis

Values are expressed as means ± SE. The unpaired Student's t-test was used when two variables were compared. When more than two variables were present, group means were compared using ANOVA followed by Fisher's protected least-significant differences test. If variances were not found to meet equality criteria, variables were compared in groups of two using Student's t-test. Differences were regarded as significant when P < 0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Acute Pancreatitis

At 6 h postinduction, vitamin E treatment did not appear to affect the severity of acute pancreatitis in Cr + CsA rats, as assessed by pancreatic water content (78.2 ± 1 vs. 76.5 ± 1% water content; P = 0.4), amylasemia (21 ± 2 vs. 18 ± 2 U/ml; P = 0.3), or pancreatic myeloperoxidase activity (290 ± 20 vs. 270 ± 22 mU/mg protein; P = 0.3).

Plasma Vitamin E

CsA treatment in rats with pancreatitis induced an increment in total plasma lipids at the end of the observation period (503 ± 27 mg/dl) compared with control rats (306 ± 37; P < 0.05). Vitamin E treatment in Cr + CsA rats normalized plasma lipid concentration (272 ± 37). No effect on plasma lipids was observed either after CsA treatment alone (261 ± 10) or after Cr pancreatitis (247 ± 10). According to these findings, plasma concentrations of vitamin E, TBARS, and 8-isoprostanes were adjusted to total lipid concentration. Two weeks after the first pancreatitis induction, vitamin E concentration was reduced in Cr pancreatitis with or without CsA treatment (Fig. 1). Vitamin E supplementation resulted in a large increment in plasma vitamin E concentration at the end of the experimental period.



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Fig. 1. Vitamin E (VitE) plasma levels at the end of the experimental period. Plasma VitE was reduced in rats with caerulein (Cr) pancreatitis. VitE supplementation resulted in a marked increase in plasma levels of VitE. Each bar represents the mean concentration (±SE) of 10 animals in each group at the end of the experimental period. *P < 0.05 vs. control. #P < 0.05 vs. all other groups. Experimental groups: control: control rats; CsA: cyclosporin A; daily treatment to control rats; Cr: rats with 2 pancreatitis inductions by Cr at day 15; Cr + CsA: CsA daily treatment to Cr rats; Cr + CsA + Vit E: VitE administered to Cr + CsA rats.

 
Effects of Vitamin E on Plasma Lipoperoxidation

Two weeks of CsA treatment to rats, with or without Cr pancreatitis, resulted in increased lipoperoxidation in blood, as demonstrated by the high levels of TBARS in plasma (Fig. 2). Vitamin E treatment in Cr + CsA rats fully prevented the increment in plasma TBARS, indicating an efficacious antioxidant effect of vitamin E counteracting CsA-enhanced oxidative stress. Measurement of 8-isoprostane is now accepted as a reliable method for the assessment of oxidative stress in vivo (32). Results on lipid peroxidation were fully confirmed by measuring 8-isoprostane concentration (Fig. 2). It is noteworthy that CsA treatment alone induced large increments in these two parameters indicative of lipid peroxidation.



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Fig. 2. Effect of VitE on 2 parameters indicative of lipid peroxidation in plasma: thiobarbituric acid-reactive substance (TBARS; A) and 8-isoprostanes (B). VitE treatment to Cr + CsA rats prevented the persistent rise in lipid peroxidation induced by CsA. Each bar represents the mean concentration (±SE) of 10 animals in each group at the end of the experimental period. *P < 0.05 vs. control, Cr, and Cr + CsA + VitE groups (A). *P < 0.05 vs. control and Cr + CsA + VitE (B). Experimental groups as in Fig. 1.

 
Effects of Vitamin E on Lipoperoxidation in Pancreatic Tissue Samples

Lipoperoxidation in pancreatic tissue, as measured by the TBARS assay, paralleled the findings observed in plasma, except that the impact of CsA treatment alone on pancreatic TBARS concentration was not as marked (Fig. 3). Treatment with CsA to rats with Cr pancreatitis resulted in a large increment in pancreatic TBARS concentration that was largely prevented by vitamin E supplementation. In addition, pancreas from rats with Cr pancreatitis showed a moderate increase in TBARS concentration compared with control rats, probably reflecting residual ongoing inflammation and tissue remodeling 7 days after the last pancreatitis induction.



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Fig. 3. Effect of VitE on pancreatic lipoperoxidation. VitE largely prevented the increase in pancreatic TBARS induced by CsA treatment to Cr pancreatitis rats. Bars represent means ± SE (n = 10). *P < 0.05 vs. all other groups. #P < 0.05 vs. control. Experimental groups as in Fig. 1.

 
Effects of Vitamin E on the Profibrogenic Activity of CsA

As we previously reported (53), plasma TGF-{beta} was persistently elevated in rats with Cr pancreatitis receiving CsA. In addition to its antioxidant effect, vitamin E treatment was also associated with a reduction in plasma TGF-{beta} (Fig. 4). In addition to its effects on plasma lipoperoxidation, CsA treatment to rats without pancreatitis was also associated with increased plasma TGF-{beta} concentration.



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Fig. 4. Effect of VitE on plasma transforming growth factor (TGF)-{beta} concentration. VitE treatment counteracted the effect of CsA on plasma TGF-{beta}. Bars represent means ± SE (n = 10). Significant difference from control, Cr, and Cr + CsA + VitE groups (*P < 0.05). Experimental groups as in Fig. 1.

 
Collagen pancreatic content, as measured by hydroxyproline concentration in pancreas, was increased in Cr + CsA rats compared with control or Cr rats (Fig. 5). Vitamin E significantly reduced the increment in collagen deposition induced by CsA in Cr pancreatitis rats. Moreover, vitamin E treatment also reduced hyaluronic acid concentration in blood (Fig. 5). Thus vitamin E effectively reduced two biochemical parameters indicative of increased ECM turnover: hydroxyproline, measured as an index of pancreatic collagen content (the representative fibrillar ECM protein), and hyaluronic acid, a soluble nonprotein element of the ECM. However, at this time point (2 wk after the first pancreatitis), vitamin E was unable to fully prevent CsA-induced fibrogenesis, because hydroxyproline and hyaluronic acid were still elevated in Cr + CsA + VitE rats compared with the Cr group.



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Fig. 5. Effect of VitE on pancreatic hydroxyproline content (A) and plasma hyaluronic acid (B), determined as 2 distinct parameters of extracellular matrix turnover. VitE greatly reversed the effects of CsA on extracellular matrix turnover in the pancreas recovering from Cr pancreatitis. Bars represent means ± SE (n = 10). Significant difference from all other groups (*P < 0.05). Experimental groups as in Fig. 1.

 
Pancreatic Morphology

Total pancreatic weight is a simple but reliable method for estimating the degree of pancreatic regeneration after injury. Vitamin E administration greatly ameliorated the pancreatic weight loss induced by CsA in Cr pancreatitis rats. However, in agreement with hydroxyproline and hyaluronic acid findings, pancreatic weight of vitamin E-treated rats was still far below the weight observed in control or Cr rats (Fig. 6).



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Fig. 6. Total pancreatic weight 2 wk after the first pancreatitis induction. Pancreatic weight was determined as an index of the degree of pancreatic recovery after injury. Bars represent means ± SE (n = 10). Significant difference from all other groups (*P < 0.05). Experimental groups as in Fig. 1.

 
Vitamin E clearly enhanced pancreatic regeneration in Cr + CsA + VitE rats, as demonstrated by light microscopy inspection (Fig. 7). The degree of acinar atrophy and the amount of pancreatic connective tissue were greatly reduced by vitamin E administration. As previously reported (53), CsA administration alone did not produce significant effects on pancreatic structure, except for minor modifications such as cytoplasm vacuolization in some acinar cells.



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Fig. 7. Representative microscopic findings of pancreatic specimens 2 wk after the first pancreatitis induction. A: Cr + CsA rats. B: Cr + CsA rats on VitE treatment. Pancreases from Cr + CsA rats show extensive loss of acinar cells with expanded connective tissue. These features are prevented in part by VitE treatment (hematoxylin and eosin staining; original magnification: x200).

 
Blind semiquantitative assessment confirmed these observations. Connective tissue accumulation was decreased by vitamin E treatment compared with Cr + CsA rats (1.2 ± 0.1 vs. 2.2 ± 0.2; P < 0.05, n = 10), and the degree of acinar atrophy was also improved (1.8 ± 0.2 vs. 2.5 ± 0.2; P < 0.05, n = 10). Immunostaining for SMAc was used to detect and quantify activated myofibroblasts. Compared with Cr + CsA rats, vitamin E treatment was associated with a reduction in the number of activated myofibroblasts present in the pancreas (29.5 ± 2.4 vs. 43.2 ± 6 cells per high-power field; P < 0.05). The distribution of SMAc within the pancreas sections was variable. SMAc were observed in clusters in areas of connective tissue expansion, whereas they kept a periacinar distribution in areas showing better-preserved architecture (Fig. 8).



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Fig. 8. Interstitial {alpha}-smooth muscle actin ({alpha}SMA) positive cells as seen after anti-{alpha}SMA immunostaining. A: Cr + CsA rats. B: Cr + CsA rats on VitE treatment. A lesser number of {alpha}SMA positive cells and a reduced intensity of staining was observed in Cr + CsA rats on VitE treatment compared with Cr + CsA rats. Interstitial {alpha}SMA positive cells were identified either in a periacinar disposition or grouped in clusters in areas of connective tissue accumulation (original magnification: x400).

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The present study suggests that disruption of pancreatic repair induced by CsA treatment is related to increased oxidative stress. Vitamin E reduces oxidative stress and greatly reverses ECM deposition and acinar cell atrophy.

The pathogenesis of chronic pancreatitis is not fully understood. There is growing recognition that persistent oxidative stress may play a role in the development and maintenance of the disease. Several lines of evidence arising from clinical settings and experimental animal models reinforce this argument (35, 36, 54).

ROS are normally produced in cells in a number of biological processes where they regulate critical physiological and pathophysiological functions (18). Elements engaged in a complex molecular machinery that prevent ROS from becoming harmful are termed endogenous antioxidants. Oxidative stress is the molecular and cellular damage resulting from excessive ROS production or from reduced endogenous antioxidants.

Assessment of oxidative stress in vivo usually relies on the determination of stable oxidatively modified molecules (17). However, these molecules may be generated in too low amounts or may be metabolized too quickly to permit reliable measurements. In our experimental model, the presence of increased plasma and pancreatic TBARS as well as circulating 8-isoprostanes several days after the last pancreatitis induction clearly indicates that CsA induces persistent lipoperoxidation. Vitamin E reduces both TBARS and 8-isoprostanes. In addition, we have shown that 8-isoprostanes (bioactive no enzymatic generated prostaglandin-like compounds derived from ROS-catalyzed peroxidation of arachidonic acid) (32) are produced in response to CsA treatment. In rats with pancreatitis, an excess of CsA-induced oxidized bioactive lipids such as 8-isoprostanes may contribute to generating further damage to the pancreas.

Administration of CsA to patients has been shown to increase plasma lipids (5, 20). Interestingly, in our experimental model, CsA treatment in rats with Cr pancreatitis was also associated with a marked rise in plasma lipids. On the contrary, total plasma lipid concentration in normal rats was not modified by CsA treatment, despite its effects on plasma lipoperoxydation. Furthermore, vitamin E supplementation to Cr + CsA rats reverted the increment in plasma lipids induced by CsA.

We have also shown that CsA treatment in Cr pancreatitis induces persistent increments in TGF-{beta} bioactivity, which are again reversed by vitamin E treatment. TGF-{beta} involvement in fibrogenesis is widely documented (55). In Cr pancreatitis, TGF-{beta} infusion enhances matrix accumulation (52), whereas antibodies raised against TGF-{beta} tend to diminish it (37). Previous studies (25) reported CsA-induced release of TGF-{beta} in the kidney and in supernatants of cultured tubule cells. On the other hand, vitamin E abrogates TGF-{beta} secretion induced by angiotensin II or thromboxane in glomerular mesangial cells (49). In the liver, vitamin E supplementation inhibits TGF-{beta} gene expression (41). In an in vivo model of pulmonary fibrosis, recent observations that provide further support to our work indicate that vitamin E supplementation decreases TGF-{beta} gene expression and pulmonary hydroxyproline content (7). Oxidative stress may either be the cause or the result of TGF-{beta} activity (11, 49). Both TGF-{beta} and oxidative stress are independently able to activate pancreatic stellate cells (3, 4, 38), manifested by "de novo" {alpha}SMA expression and by phenotypic transdifferentiation into fibroblast-like cells termed myofibroblasts. These same signals, oxidative stress and TGF-{beta}, induce apoptosis in epithelial cells that results in parenchymal atrophy (15). Thus the histological features induced by CsA treatment in our model (increased ECM, infiltrating fibroblasts, and parenchymal atrophy) tantalizingly resemble the characteristic picture of chronic pancreatitis.

Vitamin E was also shown to reduce CsA-induced lipoperoxidation, TGF-{beta} bioactivity, and the number of activated myofibroblasts. These effects run parallel to a reduction in ECM deposition and acinar cell atrophy. Vitamin E, the most abundant lipid-soluble antioxidant, is present in relevant amounts in biological membranes, being positioned as a first-line defense element against oxidative attack by ROS. {alpha}-Tocopherol concentration rises as the daily amount of vitamin E in the diet increases (19, 31). In addition, the concentration of vitamin E in mitochondrial membranes is inversely related to the production of oxidative radicals (31). It is of particular interest to note that one of the targets of CsA is, precisely, the mitochondrial membrane, a major source of ROS in nonphagocytic cells and where vitamin E displays its full antioxidant function. Not surprisingly, quite a number of studies has reported on the benefits of vitamin E supplementation on CsA-induced cytotoxicity (2, 42, 56).

Vitamin E reduced the number of myofibroblasts in the repairing pancreas, an effect also observed in other fibrogenic conditions (6). Pancreatic myofibroblast activity is thought to be the cause of increased ECM in chronic pancreatitis. However, myofibroblasts participate in normal repair processes aimed at fully restoring pancreas architecture after injury. CsA may exert its action on a number of cell types (endothelial, vascular smooth muscle, epithelial, and acinar cells) that might subsequently affect myofibroblast activity. In addition, direct effects of CsA on pancreatic myofibroblasts should also be considered. One possibility is that CsA-induced persistent increments in ROS, TGF-{beta}, or both may keep myofibroblasts in their activated profibrogenic state. Moreover, CsA inhibition of mitochondrial-induced apoptosis may prevent activated myofibroblasts from being removed from the healing tissue. Excess of matrix synthesis by redundant spared myofibroblasts might impair the necessary tissue remodeling that precedes full restoration (22). By counteracting CsA-induced oxidative stress and TGF-{beta} bioactivity, vitamin E may reverse this trend and facilitate myofibroblast apoptosis. The pathways regulated by vitamin E that are able to modulate pancreatic fibrogenesis merit further investigations.


    GRANT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
This work was supported in part by Grant FIS 01/1401 and Generalitat de Catalunya 2001SGR 00389.


    ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Pilar Chacón and María Isabel Guilera for excellent technical work in the determination of vitamin E concentrations.


    FOOTNOTES
 

Address for reprint requests and other correspondence: X. Molero, Servei d'Aparell Digestiu, Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron 119–129, 08035 Barcelona, Spain (E-mail: xmolero{at}vhebron.net).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 

  1. Abdu KM, Rao G, Montgomery CA, Dinowitz M, and Kanagalingam K. Thirteen-week toxicity study of D-alpha-tocopheryl acetate (vitamin E) in Fisher 344 rats. Food Chem Toxicol 24: 1043–1050, 1986.[CrossRef][ISI][Medline]
  2. Andres D and Cascales M. Novel mechanism of vitamin E protection against cyclosporine A cytotoxicity in cultured rat hepatocytes. Biochem Pharmacol 64: 267–276, 2002.[CrossRef][ISI][Medline]
  3. Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, and Wilson JS. Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis. Gut 44: 534–541, 1999.[Abstract/Free Full Text]
  4. Apte MV, Phillips PA, Fahmi RG, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, Naidoo D, and Wilson J. Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells. Gastroenterology 118: 780–794, 2000.[ISI][Medline]
  5. Ballantyne CM, Podet EJ, Patsch WP, Harati Y, Appel V, Gotto AM Jr, and Young JB. Effects of cyclosporine therapy on plasma lipoprotein levels. JAMA 262: 53–56, 1989.[Abstract]
  6. Brown KE, Poulos JE, Li L, Soweid AM, Ramm GA, O'Neill R, Britton RS, and Bacon BR. Effect of vitamin E supplementation on hepatic fibrogenesis in chronic dietary iron overload. Am J Physiol Gastrointest Liver Physiol 272: G116–G123, 1997.[Abstract/Free Full Text]
  7. Card JW, Racz WJ, Brien JF, and Massey TE. Attenuation of amiodarone-induced pulmonary fibrosis by vitamin E is associated with suppression of transforming growth factor-{beta}1 gene expression but not prevention of mitochondrial dysfunction. J Pharmacol Exp Ther 304: 277–283, 2003.[Abstract/Free Full Text]
  8. Chow CK, Ibrahim W, Wei Z, and Chan AC. Vitamin E regulates mitochondrial hydrogen peroxide generation. Free Radic Biol Med 27: 580–587, 1999.[CrossRef][ISI][Medline]
  9. Constantinescu A, Han D, and Packer L. Vitamin E recycling in human erythrocyte membranes. J Biol Chem 268: 10906–10913, 1993.[Abstract/Free Full Text]
  10. Crompton M, Virji S, Doyle V, Johnson N, and Ward JM. The mitochondrial permeability transition pore. Biochem Soc Symp 66: 167–179, 1999.[Medline]
  11. De Bleser PJ, Xu G, Rombouts K, Rogiers V, and Geerts A. Glutathione levels discriminate between oxidative stress and transforming growth factor-beta signaling in activated rat hepatic stellate cells. J Biol Chem 274: 33881–33887, 1999.[Abstract/Free Full Text]
  12. Demols A, Van Laethem JL, Quertinmont E, Degraef C, Delhaye M, Geerts A, and Desviere J. Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis. Am J Physiol Gastrointest Liver Physiol 282: G1105–G1112, 2002.[Abstract/Free Full Text]
  13. Elsasser HP, Adler G, and Kern HF. Time course and cellular source of pancreatic regeneration following acute pancreatitis in the rat. Pancreas 1: 421–429, 1986.[Medline]
  14. Esposito C, Fornoni A, Cornacchia F, Bellotti N, Fasoli G, Foschi A, Mazzucchelli I, Mazzullo T, Semeraro L, and Dal Canton A. Cyclosporine induces different responses in human epithelial, endothelial and fibroblast cell cultures. Kidney Int 58: 123–130, 2000.[CrossRef][ISI][Medline]
  15. Goldkorn T, Balaban N, Shannon M, Chea V, Matsukuma K, Gilchrist D, Wang H, and Chan C. H2O2 acts on cellular membranes to generate ceramide signaling and initiate apoptosis in tracheobronchial epithelial cells. J Cell Sci 111: 3209–3220, 1998.[Abstract/Free Full Text]
  16. Guechot J, Loria A, Serfaty L, Giral P, Giboudeau J, and Poupon R. Serum hyaluronan as a marker of liver fibrosis in chronic viral hepatitis C: effect of {alpha}-interferon therapy. J Hepatol 22: 22–26, 1995.[CrossRef][ISI][Medline]
  17. Gutteridge JM and Halliwell B. The measurement and mechanism of lipid peroxidation in biological systems. Trends Biochem Sci 15: 129–135, 1990.[CrossRef][ISI][Medline]
  18. Hoidal JR. Reactive oxygen species and cell signaling. Am J Respir Cell Mol Biol 25: 661–663, 2001.[Free Full Text]
  19. Ibrahim WH, Bhagavan HN, Chopra RK, and Chow CK. Dietary coenzyme Q10 and vitamin E alter the status of these compounds in rat tissues and mitochondria. J Nutr 130: 2343–2348, 2000.[Abstract/Free Full Text]
  20. Ichimaru N, Takahara S, Kokado Y, Wang JD, Hatori M, Kameoka H, Inoue T, and Okuyama A. Changes in lipid metabolism and effect of simvastatin in renal transplant recipients induced by cyclosporine or tacrolimus. Atherosclerosis 158: 417–423, 2001.[CrossRef][ISI][Medline]
  21. Inan C, Kilic I, Kilic K, Kalayci O, and Kotiloglu E. The effect of high dose antenatal vitamin E on hypoxia-induced changes in newborn rats. Pediatr Res 38: 685–689, 1995.[Abstract]
  22. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, and Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest 102: 538–549, 1998.[Abstract/Free Full Text]
  23. Israel K, Yu W, Sanders BG, and Kline K. Vitamin E succinate induces apoptosis in human prostate cancer cells: role of Fas in vitamin E succinate-triggered apoptosis. Nutr Cancer 36: 90–100, 2000.[ISI][Medline]
  24. Johnson DW, Saunders HJ, Johnson FJ, Huq SO, Field MJ, and Pollock CA. Fibrogenic effects of cyclosporin A on the tubulointerstitium: role of cytokines and growth factors. Exp Nephrol 7: 470–478, 1999.[CrossRef][ISI][Medline]
  25. Johnson DW, Saunders HJ, Johnson FJ, Huq SO, Field MJ, and Pollock CA. Cyclosporin exerts a direct fibrogenic effect on human tubulointerstitial cells: roles of insulin-like growth factor I, transforming growth factor {beta}1, and platelet-derived growth factor. J Pharmacol Exp Ther 289: 535–542, 1999.[Abstract/Free Full Text]
  26. Kanno T, Utsumi T, Takehara Y, Ide A, Akiyama J, Yoshioka T, Horton AA, Utsumi K. Inhibition of nutrophil-superoxide generation by alpha-tocopherol and coenzyme Q. Free Radic Res 24: 281–286, 1996.[ISI][Medline]
  27. Klee CB, Ren H, and Wang X. Regulation of the calmodulin-stimulated protein phosphatase, calcineurin. J Biol Chem 273: 13367–13370, 1998.[Free Full Text]
  28. Kopp JB and Klotman PE. Cellular and molecular mechanisms of cyclosporin nephrotoxicity. J Am Soc Nephrol 1: 162–179, 1990.[Abstract]
  29. Krauskopf A, Buetler TM, Nguyen NS, Mace K, and Ruegg UT. Cyclosporin A-induced free radical generation is not mediated by cytochrome P-450. Br J Pharmacol 135: 977–986, 2002.[Abstract/Free Full Text]
  30. Kropf J, Schurek JO, Wollner A, and Gressner AM. Immunological measurement of transforming growth factor-beta I (TGF-{beta}1) in blood; assay development and comparison. Clin Chem 43: 1965–1974, 1997.[Abstract/Free Full Text]
  31. Lass A and Sohal RS. Effect of coenzyme Q10 and {alpha}-tocopherol content of mitochondria on the production of superoxide anion radicals. FASEB J 14: 87–94, 2000.[Abstract/Free Full Text]
  32. Lawson JA, Rokach J, and FitzGerald GA. Isoprostanes: formation, analysis and use as indices of lipid peroxidation in vivo. J Biol Chem 274: 24441–24444, 1999.[Free Full Text]
  33. Lohr M, Fischer B, Weber H, Emmrich J, Nizze H, Liebe S, and Kloppel G. Release of hyaluronan and laminin into pancreatic secretions. Digestion 60: 48–55, 1999.[CrossRef][ISI][Medline]
  34. Mansouri A, Demeilliers C, Amsellem S, Pessayre D, and Fromenty B. Acute ethanol administration oxidatively damages and depletes mitochondrial DNA in mouse liver, brain, heart, and skeletal muscles: protective effects of antioxidants. J Pharmacol Exp Ther 298: 737–743, 2001.[Abstract/Free Full Text]
  35. Mathew P, Wyllie R, Van Lente F, Steffen RM, and Kay MH. Antioxidants in hereditary pancreatitis. Am J Gastroenterol 91: 1558–1562, 1996.[ISI][Medline]
  36. Matsumura N, Ochi K, Ichimura M, Mizushima T, Harada H, and Harada M. Study on free radicals and pancreatic fibrosis–pancreatic fibrosis induced by repeated injections of superoxide dismutase inhibitor. Pancreas 22: 53–57, 2001.[CrossRef][ISI][Medline]
  37. Menke A, Yamaguchi H, Gress TM, and Adler G. Extracellular matrix is reduced by inhibition of transforming growth factor beta1 in pancreatitis in the rat. Gastroenterology 113: 295–303, 1997.[ISI][Medline]
  38. Nieto N, Friedman SL, and Cederbaum AI. Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2E1-derived reactive oxygen species. Hepatology 35: 62–73, 2002.[CrossRef][ISI][Medline]
  39. Oberti F, Valsesia E, Pilette C, Rousselet MC, Bedossa P, Aube C, Gallois Y, Rifflet H, Maiga MY, Penneau-Fontbonne D, and Cales P. Noninvasive diagnosis of hepatic fibrosis or cirrhosis. Gastroenterology 113: 1609–1616, 1997.[ISI][Medline]
  40. Packer L, Weber SU, and Rimbach G. Molecular aspects of alpha-tocotrienol antioxidant action and cell signalling. J Nutr 131: 369S–373S, 2001.[Abstract/Free Full Text]
  41. Parola M, Muraca R, Dianzani I, Barrera G, Leonarduzzi G, Bendinelli P, Piccoletti R, and Poli G. Vitamin E dietary supplementation inhibits transforming growth factor beta 1 gene expression in the rat liver. FEBS Lett 308: 267–270, 1992.[CrossRef][ISI][Medline]
  42. Perez de Lema G, Arribas I, Prieto A, de Arriba G, Rodríguez-Puyal D, and Rodríguez Puyol M. Cyclosporin A-induced hydrogen peroxide synthesis by cultured human mesangial cells is blocked by exogenous antioxidants. Life Sci 62: 1745–1753, 1998.[CrossRef][ISI][Medline]
  43. Ricciarelli R, Maroni P, Ozer N, Zingg J, and Azzi A. Age-dependent increase of collagenase expression can be reduced by alpha-tocopherol via protein kinase C inhibition. Free Radic Biol Med 27: 729–737, 1999.[CrossRef][ISI][Medline]
  44. Riesle E, Friess H, Zhao L, Wagner M, Uhl W, Baczako K, Korc M, and Buchler MW. Increased expression of transforming growth factor beta after acute oedematous pancreatitis in rats suggests a role in pancreatic repair. Gut 40: 73–79, 1997.[Abstract]
  45. Rojkind M and Gonzalez E. An improved method for determining specific radioactivities of 14C-proline and 14C-hydroxyproline in collagen and non-collagen proteins. Anal Biochem 57: 1–7, 1974.[ISI][Medline]
  46. Rusnak F and Mertz P. Calcineurin: form and function. Physiol Rev 80: 1483–1521, 2000.[Abstract/Free Full Text]
  47. Salaris SC and Babbs CF. A rapid, widely applicable screen for drugs that suppress free radical formation in ischemia/reperfusion. J Pharmacol Methods 20: 335–345, 1988.[CrossRef][ISI][Medline]
  48. Sparmann G, Merkord J, Jaschke A, Nizze H, Jonas L, Lohr M, Liebe S, and Emmrich J. Pancreatic fibrosis in experimental pancreatitis induced by dibutyltin dichloride. Gastroenterology 112: 1664–1672, 1997.[ISI][Medline]
  49. Studer RK, Craven PA, and DeRubertis FR. Antioxidant inhibition of protein kinase C-signaled increases in transforming growth factor-beta in mesangial cells. Metabolism 46: 918–925, 1997.[ISI][Medline]
  50. Thomas SE, Andoh TF, Pichler RH, Shankland SJ, Couser WG, Bennett WM, and Johnson RJ. Accelerated apoptosis characterizes cyclosporine-associated interstitial fibrosis. Kidney Int 53: 897–908, 1998.[CrossRef][ISI][Medline]
  51. Van Dam PS, Bravenboer B, van Asbeck BS, Marx JJ, and Gispen WH. High rat food vitamin E content improves nerve function in streptozotocin-diabetic rats. Eur J Pharmacol 376: 217–222, 1999.[CrossRef][ISI][Medline]
  52. Van Laethem JL, Robberecht P, Resibois A, and Deviere J. Transforming growth factor beta promotes development of fibrosis after repeated courses of acute pancreatitis in mice. Gastroenterology 110: 576–582, 1996.[ISI][Medline]
  53. Vaquero V, Molero X, Tian X, Salas A, and Malagelada JR. Myofibroblast proliferation, fibrosis, and defective pancreatic repair induced by cyclosporin in rats. Gut 45: 269–277, 1999.[Abstract/Free Full Text]
  54. Wallig MA. Xenobiotic metabolism, oxidant stress and chronic pancreatitis. Focus on glutathione. Digestion 59, Suppl 4: 13–24, 1998.
  55. Wells RG. Fibrogenesis. V. TGF-{beta} signaling pathways. Am J Physiol Gastrointest Liver Physiol 279: G845–G850, 2000.[Abstract/Free Full Text]
  56. Wolf A, Trendelenburg CF, Díez-Fernández C, Prieto P, Houy S, Trommer WE, and Cordier A. Cyclosporin A-induced oxidative stress in rat hepatocytes. J Pharmacol Exp Ther 280: 1328–1334, 1997.[Abstract/Free Full Text]
  57. Yamada T, Sartor RB, Marshall S, Specian RD, and Grisham MB. Mucosal injury and inflammation in a model of chronic granulomatous colitis in rats. Gastroenterology 104: 759–771, 1993.[ISI][Medline]
  58. Yu W, Heim K, Qian M, Simmons-Menchaca M, Sanders BG, and Kline K. Evidence for role of transforming growth factor-beta in RRR-alpha-tocopheryl succinate-induced apoptosis of human MDA-MB-435 breast cancer cells. Nutr Cancer 27: 267–278, 1997.[ISI][Medline]
  59. Zhang JG, Nicholls-Grzemski FA, Tirmenstein MA, and Fariss MW. Vitamin E succinate protects hepatocytes against the toxic effect of reactive oxygen species generated at mitochondrial complexes I and III by alkylating agents. Chem Biol Interact 138: 267–284, 2001.[CrossRef][ISI][Medline]
  60. Zingarelli B, Szabó C, and Salzman AL. Reduced oxidative and nitrosative damage in murine experimental colitis in the absence of inducible nitric oxide synthase. Gut 45: 199–209, 1999.[Abstract/Free Full Text]




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