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BASIC–LIVER, PANCREAS, AND BILIARY TRACT

Angiotensin-Converting Enzyme Inhibitor Attenuates PancreaticInflammation and Fibrosis in Male Wistar Bonn/Kobori Rats

ATSUSHI KUNO,* TAMAKI YAMADA,* KAZUHIKO MASUDA,‡ KUMIKO OGAWA,§ MITSUE SOGAWA,�

SOICHI NAKAMURA,* TAKAHIRO NAKAZAWA,* HIROTAKA OHARA,* TOMOYUKI NOMURA,*TAKASHI JOH,* TOMOYUKI SHIRAI,§ and MAKOTO ITOH**First Department of Internal Medicine, ‡First Department of Surgery, and §First Department of Pathology, Nagoya City University MedicalSchool, Nagoya, Aichi; �Third Department of Internal Medicine, Osaka City University Medical School, Osaka, Osaka, Japan

Background & Aims: Pancreatic stellate cells have somesimilarities to hepatic stellate cells and an intrinsic re-nin-angiotensin system is present in the pancreas and isenhanced in acute pancreatitis and chronic pancreatichypoxia. We assessed the effects of lisinopril, an angio-tensin-converting enzyme (ACE) inhibitor, on spontane-ously occurring chronic pancreatitis. Methods: Lisinoprilin drinking water (20, 50, or 200 mg/L) was adminis-tered to 10-week-old male Wistar Bonn/Kobori (WBN/Kob) rats for 10 weeks and then the inflammatory pa-rameters, fibrosis, serum and pancreatic ACE activity,and expression of transforming growth factor-�1 (TGF-�1) messenger RNA (mRNA) as well as positive immu-nostaining for �-smooth muscle actin (�-SMA) were as-sessed. Results: Lisinopril attenuated gross alterationsin the pancreas. This protective effect was confirmedquantitatively by significant increases in pancreaticweights and decreases in pancreatic myeloperoxidase(MPO) activity (an index of granulocyte infiltration), pan-creatic hydroxyproline content (an index of collagen dep-osition), ratio of fibrous tissue, and histologic scores.Lisinopril significantly reduced serum ACE activity but itdid not affect pancreatic activity. High doses of lisinoprilsuppressed the overexpression of TGF-�1 mRNA mea-sured by reverse-transcription polymerase chain reac-tion (RT-PCR) and decreased the number of �-SMA–positive cells (activated pancreatic stellate cells) in thepancreas. Conclusions: Lisinopril alleviated chronic pan-creatitis and fibrosis in male WBN/Kob rats. It sup-pressed the expression of TGF-�1 mRNA, resulting in theprevention of pancreatic stellate cell activation, whichmay be involved in the observed protection. We proposethat an ACE inhibitor may be useful for treating chronicpancreatitis.

Chronic pancreatitis is an irreversible progressive dis-ease characterized by destruction of acinar and duc-

tal cells. Increased accumulation of extracellular matrix isa histologic characteristic of chronic pancreatitis that

results in pancreatic fibrosis. The lack of an easily repro-ducible animal model has limited our understanding ofthe pathogenetic mechanisms responsible for chronicpancreatitis and pancreatic fibrosis. Recently, we focusedon the spontaneously occurring chronic pancreatitis andsubsequent diabetes in male Wistar Bonn/Kobori(WBN/Kob) rats.1–3 These rats completely reproducechronic pancreatitis, which is accompanied by parenchy-mal destruction and its replacement with fibrosis, result-ing in both endocrine and exocrine dysfunction.1–3 Weshowed that apoptosis of acinar cells contributes to thedevelopment of chronic pancreatitis and that T cells areinvolved in acinar cell apoptosis and the pathogenesis ofchronic pancreatitis.4,5 However, the factors involved inpancreatic inflammation and fibrosis remain unclear inthis model. Su et al.6 reported that the level of trans-forming growth factor (TGF)-�1 messenger RNA(mRNA) peaked at 12 weeks when acute pancreatitis wasobserved and that TGF-�1 expression may be a trigger ofpancreatic fibrosis in these rats.

TGF-�1 is a multifunctional cytokine7 that mediatesfibrosis in heart diseases, glomerular diseases, idiopathicpulmonary fibrosis, and liver diseases.8–14 TGF-�1mRNA and protein are localized in pancreatic acinar andductal cells15,16 and TGF-�1 precursor and its latentbinding protein are present in the inflamed lesions withfibrosis in human chronic pancreatitis.17 TGF-� pro-motes the development of pancreatic fibrosis after recur-

Abbreviations used in this paper: ACE, angiotensin-converting en-zyme; �-SMA, �-smooth muscle actin; AT, angiotensin; HSC, hepaticstellate cells; MPO, myeloperoxidase; PSC, pancreatic stellate cells;RAS, renin angiotensin system; RT-PCR, reverse-transcription polymer-ase chain reaction; TGF, transforming growth factor; WBN/Kob, WistarBonn/Kobori.

© 2003 by the American Gastroenterological Association0016-5085/03/$30.00

doi:10.1053/gast.2003.50147

GASTROENTEROLOGY 2003;124:1010–1019

rent episodes of acute pancreatitis in mice,18 and trans-genic mice overexpressing TGF-�1 in the pancreasdevelop pancreatic fibrosis and destruction of exocrinepancreas.19 In addition, inhibition of TGF-�1 results inreduction of extracellular matrix protein in the rat.20

These findings support the idea that TGF-�1 plays animportant role in pancreatic fibrosis. Thus, a procedurefor suppressing TGF-�1 may be useful for treating pan-creatic fibrosis.

Angiotensin (AT)-II, an octapeptide produced by pro-teolytic cleavage of its precursor AT-I by angiotensin-converting enzyme (ACE), induces contraction and pro-liferation of mesangial cells and hepatic stellate cells(HSCs).21,22 AT-II increases the expressions of theTGF-�1 and collagen I genes in lung fibroblasts23 andstimulates the proliferation of mesangial cells24,25 andHSCs and their synthesis of extracellular matrix proteinsthrough induction of TGF-� expression in vivo and invitro.12–14 Recently, it was shown that an ACE inhibitorsuppressed progression of hepatic fibrosis in rats.26 Inaddition, it has been reported that AT-II and AT-Ireceptor interaction results in hepatic fibrosis throughactivation of HSCs and their increased expression ofTGF-�1 in rats, which were suppressed by an AT-II–receptor antagonist in vivo and in vitro.27 These factsstrongly suggest that AT-II, converted endogenouslyfrom AT-I by ACE, plays a major role in the develop-ment of hepatic fibrosis.

Isolated pancreatic stellate cells (PSCs) have somesimilar properties to those of HSCs with regard to shape,presence of vitamin A, positive staining for desmin andglial fibrillary acidic protein, and positive staining for�-smooth muscle actin (�-SMA) in the active phase.28,29

It is well documented that PSCs are involved in thepathogenesis of pancreatic fibrosis in both experimentalanimals and humans.30,31 It has been shown that therenin-angiotensin system (RAS) is present intrinsically inthe pancreas and that its level is enhanced during acutepancreatitis and chronic pancreatic hypoxia in the exper-imental animals.32–35 Therefore, we hypothesized thatRAS may be involved in the pathogenesis of pancreaticinflammation and fibrosis and ACE inhibition may alle-viate pancreatic fibrosis by suppressing induction ofTGF-�1 and activation of PSCs, thereby reducing theirproduction of extracellular matrix proteins as observed inkidney, lung, and liver. However, there is no informationregarding the effect of ACE inhibition on pancreaticinflammation and/or fibrosis. We investigated the effectsof lisinopril, an ACE inhibitor, on pancreatic inflamma-tion and fibrosis in male WBN/Kob rats. We also as-sessed its effects on the expression of the TGF-�1 gene

and on �-SMA–positive cells (activated PSCs) in thepancreas.

Materials and MethodsMaterials and Animals

WBN/Kob and Wistar rats were purchased from SLC(Hamamatsu, Japan) and kept in a temperature-controlledroom under constant light. They were allowed free access towater and standard laboratory feed. The study protocol wasapproved by the Animal Care Committee of Nagoya CityUniversity. Lisinopril was a generous gift from Shionogi Co.,Ltd. (Osaka, Japan). All other chemicals were of the highestquality available.

Groups of Animals and Treatment

Thirty-three WBN/Kob (10-week-old) rats were di-vided randomly into the untreated group (n � 10), and low(n � 5), medium (n � 5), and high (n � 13) doses of lisinoprilgroups for determination of pancreatic myeloperoxidase(MPO) activity, pancreatic hydroxyproline content, serumACE activity, and histologic assessment, including scores andratios of fibrous tissue assessed by Azan staining. PancreaticACE activity was measured in the untreated (n � 10) andhigh-dose (n � 13) groups. In the second study, 8 rats, also 10weeks old, were divided into the untreated (n � 3) andhigh-dose lisinopril (n � 5) groups for expression of TGF-�1mRNA and immunohistochemical staining for �-SMA. Wepreviously reported that body weight and pancreas weightsignificantly decreased and pancreatic MPO activity signifi-cantly increased in 20-week-old male WBN/Kob rats com-pared with the age-matched male Wistar rats in which thepancreas was intact.4,5 In the present study, 20-week-old maleWistar rats (n � 5) were used for determination of pancreatichydroxyproline content and TGF-�1 mRNA in the pancreas.

The concentration of lisinopril in drinking water, 200mg/L, in the high-dose group was according to the previousreports 4,36,37 and its estimated dose was 20 mg � kg�1 � day�1.4

This dose of lisinopril was reported to be effective for reversingleft ventricular fibrosis and dysfunction through ACE inhibi-tion in rats.36,37 We also tested the effects of low (20 mg/L)and medium (50 mg/L) concentrations of lisinopril in drinkingwater to assess its dose-dependent effect. They were refreshedtwice a week, it was given for 10 weeks, and the amountconsumed was calculated. Body weights were recorded weekly.

Tissue Sampling

All WBN/Kob rats treated for 10 weeks were killedwith an overdose of pentobarbital sodium (Abbott Laborato-ries, North Chicago, IL). Pancreas tissues were kept at �80°Cfor determination of MPO activity, hydroxyproline content,and ACE activity or immediately placed in liquid nitrogen andkept at �80°C for assessment of the expression of TGF-�1mRNA by reverse-transcription polymerase chain reaction(RT-PCR). Their pancreases also were fixed in 10% buffered

April 2003 ACE INHIBITOR IN WBN/Kob RATS 1011

formalin or 4% paraformaldehyde in phosphate-buffered salinefor histologic assessment or immunohistochemistry, respec-tively. In addition, pancreases from 20-week-old Wistar ratswere kept at �80°C for determination of hydroxyprolinecontent or immediately placed in liquid nitrogen for RT-PCRanalysis.

Histologic Analysis

Pancreas tissue was fixed with 10% buffered formalin,processed for paraffin-embedded sections, and stained withH&E and Azan. Microscopic examination was performed by apathologist who was unaware of the groups of the rats. Thestatus of inflammation was evaluated as the grades of inflam-matory cell infiltration, interstitial edema, fibrosis, acinar cellnecrosis, and hemorrhage on a scale of negligible to maximal(0–3) as reported previously.4,5 The scores were comparedamong the untreated and low-, medium-, and high-dose lis-inopril groups. Quantity of fibrosis was analyzed with the aidof an image processor (Image Processor for Analytical Pathol-ogy; Sumika Technoservice, Osaka, Japan) as the ratio ofanilin-blue–positive fibrous tissue per total area in wholeAzan-stained pancreas sections, excluding lymph nodes andmajor vessels if present.38

Immunohistochemistry for �-SmoothMuscle Actin

Twenty-four hours after fixation at 4°C with 4% para-formaldehyde in phosphate-buffered saline, tissues were trans-ferred successively into 8% to 20% sucrose in 0.1 mol/Lphosphate buffer. The tissues were frozen in liquid nitrogenand cryosections were cut with a CM3050 cryostat (LeicaInstruments GmbH, Nussloch, Germany). To block endoge-nous peroxidase activity, sections were treated with 0.3%hydrogen peroxide in methanol. After washing with phos-phate-buffered saline, they were treated for 1 hour at roomtemperature with 5% fetal bovine serum to block nonspecificreactions. They were then incubated overnight at room tem-perature with mouse anti-human �-SMA monoclonal antibody(1:750 dilution; Dako, Carpinteria, CA), which can detect rat�-SMA.39 After washing, the sections were incubated for 1hour at room temperature with biotinylated rabbit anti-mouseimmunoglobulin G antibody (1:500 dilution; Dako) and thenfor 1 hour at room temperature with horseradish peroxidase–labeled streptavidin (LSAB kit; Dako). Reaction products werevisualized by treating sections for 3–5 minutes with 0.2mg/mL 3,3�-diaminobenzine tetrahydrochloride in 0.05 mol/LTris-buffered saline, pH 7.4, containing 0.003% hydrogenperoxide. Nuclei were counterstained with methyl green.

Measurement of PancreaticMyeloperoxidase Activity

Pancreatic MPO activity, an indirect quantitative in-dex of granulocyte infiltration, was determined using themethod of Grisham et al.40 with a minor modification. Briefly,pancreatic tissue was homogenized in 20 mmol/L phosphate

buffer (pH 7.4) and the homogenate was centrifuged at 6000� g for 20 minutes at 4°C. The pellet was homogenized andsonicated with an equivalent volume of 50 mmol/L acetic acid(pH 6.0) containing 0.5% (wt/vol) hexadecyltrimethylammo-nium hydroxide. MPO activity was determined by measuringthe H2O2-dependent oxidation of 3,3�,5,5� tetramethylbenzi-dine and is expressed as units per gram wet weight of pancreas.

Measurement of Pancreatic Hydroxyproline

Hydroxyproline content, an indicator of collagen dep-osition, was determined by a modified method of Blumen-krantz and Asobe-Hansen.41 Briefly, approximately 100 mg ofpancreas specimen was homogenized in water and hydrolyzedat 110°C for 20 hours in 10 N HCl. HCl was evaporated undernitrogen gas and the hydrolysate was dissolved in distilledwater and filtered. Then 0.5-mL aliquots were mixed with 3mL of citrate-phosphate buffer (0.15 mol/L citric acid and 0.6mol/L dibasic sodium phosphate) and 0.5 mL of 1 mol/Lperiodic acid in 9 mol/L H3PO4. The samples were then mixedwith 1.75 mL of extract buffer consisting of toluene (5 parts),2-methyl-1-propanol (5 parts), and 1-propanol (2 parts),shaken for 30 minutes, and centrifuged. The organic phase (0.6mL) was mixed with Ehrlich’s reagent (0.15 mL) and left for15 minutes. The absorbance was measured at 565 nm andhydroxyproline levels were calculated using a standard curvemade with 4-hydroxy-1-proline and expressed as microgramsper gram tissue.

Measurement of Serum Lisinopril Levels

Serum lisinopril levels were determined in the low-dose (n � 5), medium-dose (n � 5), and high-dose (n � 13)groups by using radioimmunoassay as reported previously.42

Measurement of Serum and PancreaticAngiotensin-Converting Enzyme Activity

Serum ACE activity was determined by using thecommercial kit, an ACE color (Fujirebio Co., Tokyo, Japan)and expressed as IU/L. Pancreatic ACE activity was determinedby the method of Ogiku et al.43 with a minor modification.Pancreas specimen was homogenized in 0.01 mol/L Tris HCl/0.2 mol/L sucrose (pH 7.4) on ice. Homogenate was sonicatedfor 10 seconds twice on ice and then centrifuged at 11,500 �g for 20 minutes at 4°C. Pancreatic ACE activity in thesupernatant was determined using an ACE color and expressedas nmol/mg tissue/hour.

Reverse-Transcription Polymerase ChainReaction

Total RNA was extracted from frozen pancreatic tis-sues with Trizol reagent (Invitrogen Co., Carlsbad, CA) and 2mg was reverse transcribed into complementary DNA usingOligo (dT) 12-18 Primer (Invitrogen Co.), Superscript IIRNase H-Reverse Transcriptase (Invitrogen Co.), and RNaseInhibitor (Toyobo Co., Ltd., Osaka, Japan). PCR was per-formed with reaction mixtures containing 2.5 mmol/L de-

1012 KUNO ET AL. GASTROENTEROLOGY Vol. 124, No. 4

oxynucleoside triphosphate, 10 mmol/L sense and anti-senseprimers, and 5 U/mL Taq DNA polymerase (Takara Shuzo Co.,Otsu, Japan) in a thermal cycler for 1 minute at 94°C, 1minute at 55°C, and 2 minutes at 72°C, for 32 cycles, andthen an extension reaction was performed at 72°C for 5 min-utes. The PCR primers, designed according to a previousreport,44 were: TGF-�1 sense 5�-GCGGACTACTACGC-CAAAGA-3�, anti-sense 5�-TGGTTGTAGAGGGCAAG-GAC-3�, and �-actin, sense 5�-TGGCCTCACTGTCCAC-CTTC-3�, anti-sense 5�-CGAATGGCTGACCATTCAGA-3�.PCR products were electrophoresed on a 1% agarose gel andvisualized by ethidium bromide staining. Gels were photo-graphed under ultraviolet light.

Statistics

Data are expressed as arithmetic means � SD. Statis-tical differences between 2 groups or among groups wereidentified using Student t test or one-way analysis of variance,followed by multiple comparisons using the least significancedifference method, respectively. The histologic scores andquantitative ratio of fibrous tissue were analyzed usingKruskal–Wallis rank analysis and Scheffe correction with rawscore data.

ResultsBody Weight, Dose of Lisinopril, andPancreas Weight

The body weights of 20-week-old WBN/Kob ratswere significantly lower in the medium- and high-doselisinopril groups than the untreated and low-dose groups(Table 1). The average doses of lisinopril calculated fromwater consumption and body weight were 1.8 � 0.2,5.1 � 0.5, and 21.6 � 1.8 mg � kg�1 � day�1 in the low-,medium-, and high-dose groups, respectively (Table 1).The pancreas weights increased significantly in the high-dose group compared with the other groups (Table 1).

Macroscopic Findings

As shown in Figure 1A, the pancreases in theuntreated group were severely atrophic and had widely

spread brown and red foci. Low doses of lisinopril had noeffect on macroscopic appearance. Medium doses par-tially attenuated atrophy and development of brown andred foci except in one rat in which pancreas was severelyatrophic and had colored foci. In contrast, the pancreaseshad almost intact appearances in the high-dose group(Figure 1B).

Microscopic Findings

All untreated rats had focal severe inflammationin their pancreas (Figure 2A ). Massive infiltration ofneutrophils, lymphocytes, and plasma cells, disappear-ance of acinar cells and replacement with fibrous tissue,the typical appearance of chronic active inflammation,were observed in the untreated group. Fine to thickcollagen fiber stained in blue with Azan was presentabundantly between the remaining acinar glands (Figure2B). Ductal cells and islets tended to remain longer thanacinar cells.

Interstitial edema, acinar cell necrosis, and hemor-rhage with hemosiderin extraction also were present inthe untreated group. The similar inflammatory appear-ances and slightly milder fibrosis were observed in thelow-dose group. Medium-dose treatment considerablysuppressed histologic alterations in the pancreas exceptone specimen that was associated with moderate degreesof inflammatory cell infiltration, edema, hemorrhage,acinar cell necrosis, and fibrosis. The histologic appear-ances of the pancreases in the high-dose group werealmost normal, with occasional small foci of inflamma-tory cell infiltration and fibrosis (Figure 2C ). Fine colla-gen fiber was seen occasionally between acini (Figure2D). All and most of the histologic scores were reducedsignificantly in the high- and medium-dose groups, re-spectively, compared with the untreated group, althoughthey were not reduced in the low-dose group (Table 2).The quantitative evaluation of fibrosis, indicated by theratio of fibrous tissue per total area, showed that low,

Table 1. Calculated Doses of Lisinopril and Effects of Low, Medium, and High Doses of Lisinopril in Drinking Water onBody Weight and Pancreas Weight in 20-Week-Old Male WBN/Kob Rats

Calculated dose of lisinopril(mg/kg/day)

Body weight(g)

Pancreas weight(g/kg)

Untreated (n � 10) 377.9 � 17.7 1.96 � 0.47Low (n � 5) 1.8 � 0.2 359.0 � 5.2 2.21 � 0.27Medium (n � 5) 5.1 � 0.5 333.0 � 29.8a,b 2.30 � 0.63High (n � 13) 21.6 � 1.8 330.2 � 20.3c 3.10 � 0.51d

NOTE. Statistical analyses were performed with one-way analysis of variance, followed by multiple comparisons using the least significancedifference method.a,bStatistically different from the corresponding data in the low-dose group by P � 0.05 and P � 0.01, respectively.cStatistically different from the corresponding data in the untreated and low-dose groups by P � 0.01.dStatistically different from the corresponding data in the other groups by P � 0.01.


Figure 1. Gross appearance of the pancreas in the (A) untreated and (B) high-dose lisinopril groups. (A) The pancreas is severely atrophic andcontains widely spread brown and red foci. (B) The pancreas has an almost intact appearance.

Figure 2. Representative histologic appearance of the pancreas in the untreated (A, B) and high-dose lisinopril (C, D) groups (A, C: H&E staining;B, D: Azan staining 400�). Untreated group. (A) Medium infiltration of neutrophils, lymphocytes, and plasma cells, disappearance of acinar cellsand replacement with fibrous tissue are evident. This view shows a score of 2 for inflammatory cell infiltration, edema, and fibrosis, and a scoreof 1 for acinar cell necrosis and hemorrhage. (B) Fine to thick collagen fiber is stained in blue. High-dose lisinopril group. (C ) Focal inflammatorychanges are observed slightly around the duct. This view shows a score of 1 for inflammation and fibrosis and 0 for other categories. (D) Finecollagen fiber is seen between acinar.


medium, and high doses significantly reduced fibrosis to36.1%, 28.8%, and 28.8% of the untreated group, re-spectively (Table 2).

Pancreatic Myeloperoxidase Activity

Lisinopril treatment dose-dependently reducedpancreatic MPO activity, an index of granulocyte infil-tration. The values were suppressed significantly in thehigh- and medium-dose groups compared with the othergroups (Table 3).

Pancreatic Hydroxyproline Content

Pancreatic hydroxyproline content, an indicatorof collagen deposition, significantly increased in theuntreated group compared with 20-week-old maleWistar rats (619.0 � 199.1 vs. 152.2 � 37.7 g/g,P � 0.01). Both low and medium doses of lisinopriltended to decrease hydroxyproline content (Table 3).High-dose treatment significantly suppressed hy-droxyproline content compared with the other groups(Table 3).

Serum Levels of Lisinopril

Serum lisinopril levels in the low-, medium-, andhigh-dose groups were 18.4 � 3.8, 112.0 � 58.5, and

383.8 � 197.1 ng/mL after 10 weeks of treatment,respectively.

Serum and Pancreatic Angiotensin-Converting Enzyme Activity

Lisinopril treatment dose-dependently reducedserum ACE activity and the values were significantlylower in the low-, medium-, and high-dose groups thanthe untreated group (Table 3). High doses of lisinoprildid not reduce pancreatic ACE activity compared withthe untreated group (Table 3).

Expression of Transforming GrowthFactor-�1 Messenger RNA

RT-PCR revealed that TGF-�1 mRNA was over-expressed in the pancreas in the untreated WBN/Kobrats, whereas it was only detected slightly in male Wistarrats (Figure 3). High doses of lisinopril suppressed theoverexpression of TGF-�1 mRNA in WBN/Kob rats(Figure 3).

Immunohistochemistry for �-SmoothMuscle Actin

�-SMA–positive cells that have the morphologyof activated PSCs were localized in the peri-acinar fi-

Table 2. Effects of Low, Medium, and High Doses of Lisinopril in Drinking Water on Histologic Alterations in the Pancreas in20-Week-Old Male WBN/Kob Rats

Histologic Scorea

Ratio of fibroustissue (%)

Inflammatorycell infiltration

Interstitialedema Hemorrhage

Acinar cellnecrosis Fibrosis

Untreated (n � 10) 2.2 � 0.6 2.0 � 0.7 2.3 � 0.7 0.7 � 0.7 2.3 � 0.5 37.1 � 13.9Low (n � 5) 2.0 � 0.7 2.0 � 1.0 1.8 � 0.4 1.6 � 0.5 1.8 � 0.4 13.4 � 4.9b

Medium (n � 5) 1.0 � 0.7 0.4 � 0.9c,d 1.0 � 0.7e 0.4 � 0.9f 0.6 � 0.9c 10.8 � 7.2b

High (n � 13) 1.1 � 1.0e 0.1 � 0.3b,g 0.9 � 0.9c 0.2 � 0.4d 1.0 � 0.9c 10.7 � 3.9b

aScores of each factor are indicated as averages and SDs of the grade, none, mild, moderate, and severe (0–3). Statistical analyses wereperformed with Kruskal-Wallis rank analysis and Scheffe correction with raw score data.b,c,eStatistically different from the corresponding data in the untreated group by P � 0.001, P � 0.01, and P � 0.05, respectively.d,f,gStatistically different from the corresponding data in the low-dose group by P � 0.01, P � 0.05, and P � 0.001, respectively.

Table 3. Effects of Low, Medium, and High Doses of Lisinopril in Drinking Water on Pancreatic MPO Activity, PancreaticHydroxyproline Content, and Pancreatic and Serum ACE Activity in 20-Week-Old Male WBN/Kob Rats

Pancreatic MPO activity(U/g)

Pancreatic hydroxyproline content(�/g)

ACE activity

Pancreas(nmol/mg tissue/hr)

Serum(IU/L)

Untreated (n � 10) 5.64 � 2.42 619.0 � 199.1 1.41 � 0.52 13.44 � 1.05Low (n � 5) 4.11 � 0.97 471.8 � 102.8 4.20 � 0.86a

Medium (n � 5) 3.06 � 0.80a 435.8 � 232.0 2.76 � 0.89a,b

High (n � 13) 2.26 � 1.00a,b 239.2 � 144.1a,c 1.83 � 0.55 1.40 � 0.71d

NOTE. Statistical analyses were performed with one-way analysis of variance, followed by multiple comparisons using the least significancedifference method.aStatistically different from the corresponding data in the untreated group by P � 0.01.bStatistically different from the corresponding data in the low group by P � 0.05.cStatistically different from the corresponding data in the other groups by P � 0.01.


brotic areas and vascular walls in the untreated group(Figure 4A ). In contrast, �-SMA–positive cells wereobserved only in the vascular walls in most of the spec-imens in the high-dose group (Figure 4B). �-SMA–positive cells were barely seen in the periductal areas inthe high-dose group (Figure 4C ).

DiscussionWe showed that lisinopril, an ACE inhibitor,

attenuates chronic inflammation and fibrosis in the pan-creas in male WBN/Kob rats. This protective effect wasconfirmed quantitatively by a significant increase in pan-creatic weights and decreases in pancreatic MPO activity(an index of granulocyte infiltration), pancreatic hy-droxyproline content (an index of collagen deposition),ratio of fibrous tissue, and histologic scores. We recog-nized that lisinopril reduced serum ACE activity but itdid not alter pancreatic activity. We also confirmed thathigh doses of lisinopril suppressed overexpression ofTGF-�1 mRNA and decreased the number of �-SMA–positive cells (activated PSCs) in the pancreas. We con-clude that suppressed expression of TGF-�1, which re-sults in prevention of activation of PSCs and theirsynthesis of extracellular matrix protein, may be involvedin the protective mechanism and that an ACE inhibitormay be useful for treating chronic pancreatitis.

It has been shown that autocrine and paracrine stim-ulation of HSCs by TGF-� induces activation of HSCs.12

TGF-� induces production of extracellular matrix pro-tein by HSCs and suppresses matrix degradation byinhibiting matrix metalloproteinases, resulting in the dep-osition of extracellular matrix protein in the liver.12–14

TGF-�1 also induces proliferation of PSCs and stimu-lates their synthesis of extracellular matrix protein.29–31

Therefore, we assumed that a pharmacologic down-reg-ulation of TGF-�1 production may be effective for at-tenuating pancreatic fibrosis in the present model. In-deed, treatment with high doses of lisinopril in drinking

Figure 3. Expression of TGF-�1 mRNA analyzed by RT-PCR in theuntreated and high-dose lisinopril groups and 20-week-old maleWistar rats. The �-actin mRNA is expressed in each pancreatic tissueas an internal control.

Figure 4. Immunohistochemical staining for �-SMA in the pancreas inthe (A) untreated and (B, C) high-dose lisinopril groups (�400). (A)�-SMA–positive cells with the stellate morphology (activated PSCs) werepresent in the peri-acinar fibrotic areas and vascular walls. This viewshows all scores of 1 but a score of 2 for fibrosis. (B) �-SMA–positivecells were present only in the vascular walls in most of the specimens.This view shows scores of 0 for all categories. (C) �-SMA–positive cellsbarely were observed in the periductal areas. This view shows scores of1 for fibrosis and inflammation and 0 for other categories.


water for 10 weeks suppressed the expression of TGF-�1mRNA analyzed by RT-PCR and lisinopril decreasedpancreatic collagen deposition determined by both bio-chemical and morphologic methods and decreased hy-droxyproline content and the ratio of fibrous tissue.Immunohistochemical staining revealed that high dosesof lisinopril also decreased the number of �-SMA–posi-tive cells and activated PSCs in the pancreas. We spec-ulate that suppressed expression of TGF-�1 mRNA mayprevent activation of PSCs and their synthesis of extra-cellular matrix proteins, leading to reduced pancreaticfibrosis.

It has been shown that AT-II is involved in thedevelopment of fibrosis in the heart, kidney, lung, andliver during chronic inflammation.22–25,45 An ACE in-hibitor suppressed hepatic fibrosis in the rat bile ductligation model.26 AT-II and AT-I receptor interactioncaused activation of HSCs and their increased expressionof TGF-�1, resulting in hepatic fibrosis in rats.27 Thesefindings show the important role of RAS in the devel-opment of liver fibrosis. On the other hand, the mRNAlevels of angiotensinogen and AT-II receptors are en-hanced in acute experimental pancreatitis and chronicpancreatic hypoxia.34,35 However, the role of RAS has notbeen investigated in chronic pancreatic inflammation andfibrosis. Here we presented evidence for the protectiveeffect of an ACE inhibitor. We found that high doses oflisinopril are effective for decreasing the serum ACEactivity by 89.6% although the serum level of lisinopril,383.8 � 197.1 ng/mL, is much higher than the 50%inhibitory concentration of ACE (1.3 ng/mL) in the rat.46

We further showed that lower concentrations of lisino-pril in drinking water, which achieved the serum levelsof 18.4 � 3.8 and 112.0 � 58.5 ng/mL, decreased theserum ACE activities by 68.8% and 79.5%, respectively.Despite the insufficient effects on the inflammatory pa-rameters, fibrosis was slightly milder and the ratio offibrous tissue was decreased significantly in the low-dosegroup. In the medium-dose group, although most of thehistologic scores and the ratio of fibrous tissue werereduced significantly, the pancreas weight was decreased.These data indicated that the protective effects wereinsufficient in both the low- and medium-dose groups.We speculate that more than 5 mg � kg�1 � day�1 oflisinopril, which results in higher than 100 ng/mL of itsserum level and decrease in serum ACE activity by 80%to 90%, may be necessary to suppress chronic inflamma-tion and fibrosis in the pancreas.

It is reported that chronic oral administration of lis-inopril, which achieves the plasma level of 44.0 ng/mL,effectively attenuates ACE activities of plasma and tis-

sues, including lung and kidney.47 It also is shown thatimidapril, an ACE inhibitor, improves isoproterenol-induced heart injury by suppression of the cardiac ACEactivity as well as circulating ACE activity.43 To ourknowledge, there has been no information available as topancreatic ACE activity, although ACE is detected im-munohistochemically in the vascular endothelium of thepancreas48 and the mRNA, and protein of angiotensino-gen and mRNA of AT-II receptors are identified in thepancreas.32,33 We showed that even high doses of lisino-pril did not decrease pancreatic ACE activity in maleWBN/Kob rats. Although we could not elucidate thereason for this discrepancy, it seems likely that circulat-ing ACE activity rather than pancreatic activity may beimportant in the pathogenesis of the present model.

The present findings strongly suggested that AT-IIconverted endogenously from AT-I by circulating ACEmay be involved directly in the induction of pancreaticinflammation as well as the development of pancreaticfibrosis in the present model. Because AT-II, a potentconstrictor for vascular smooth muscle cells, and itsreceptor interaction play an important role in the endo-crine regulation of pancreatic blood flow in the ro-dents49,50 and humans,51 reduced production of AT-II byACE inhibition may affect pancreatic blood flow, espe-cially in pathologic conditions. Because ischemia is spec-ulated to be involved in the pathogenesis of chronicpancreatitis in the present model,1–3 we speculated thatlisinopril may normalize pancreatic ischemia, therebyattenuating chronic inflammation and subsequent fibro-sis. However, additional studies are needed to clarifyfurther the protective mechanism.

The present study shows the efficacy of prophylacticadministration of lisinopril, an ACE inhibitor, for sup-pressing chronic inflammation and fibrosis in the pan-creas in male WBN/Kob rats and casts a new light on therole of RAS in chronic pancreatitis. Our results suggesta new strategy for the treatment of human chronic pan-creatitis. Caution is necessary, however, because ACEinhibitors occasionally induce acute human pancreaticinjury.52 Therefore, the effect of an ACE inhibitor needsbe tested in various types of chronic pancreatitis modelsbefore it is used clinically.

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Received June 8, 2002. Accepted December 20, 2002.Address requests for reprints to: Tamaki Yamada, M.D., Ph.D., First

Department of Internal Medicine, Nagoya City University MedicalSchool, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, Japan467-8601. e-mail: [email protected]; fax: (81) 52-852-0952.
