Laboratory Investigation (2010) 90, 1179–1188 & 2010 USCAP, Inc All rights reserved 0023-6837/10 $32.00

Connective tissue production by activated pancreatic stellate cells in mouse alcoholic chronic pancreatitis Alyssa L Charrier1,2 and David R Brigstock1,2,3

Alcoholic chronic pancreatitis (ACP) is characterized by pancreatic necrosis, inflammation, and scarring, the latter of which is due to excessive collagen deposition by activated pancreatic stellate cells (PSC). The aim of this study was to establish a model of ACP in mice, a species that is usually resistant to the toxic effects of alcohol, and to identify the cell type(s) responsible for production of connective tissue growth factor (CTGF), a pro-fibrotic molecule. C57Bl/6 male mice received intraperitoneal ethanol injections for 3 weeks against a background of cerulein-induced acute pancreatitis. Peak blood alcohol levels remained consistently high in ethanol-treated mice as compared with control mice. In mice receiving ethanol plus cerulein, there was increased collagen deposition as compared with other treatment groups as well as increased frequency of a-smooth muscle actin and desmin-positive PSC, which also showed significantly enhanced CTGF protein production. Expression of mRNA for collagen a1(I), a-smooth muscle actin or CTGF were all increased and co- localized exclusively to activated PSC in ACP. Pancreatic expression of mRNA for key profibrotic markers were all increased in ACP. In conclusion, a mouse model of ACP has been developed that mimics key pathophysiological features of the disease in humans and which shows that activated PSC are the principal producers of collagen and CTGF. PSC-derived CTGF is thus a candidate therapeutic target in anti-fibrotic strategies for ACP. Laboratory Investigation (2010) 90, 1179–1188; doi:10.1038/labinvest.2010.82; published online 5 April 2010

KEYWORDS: connective tissue growth factor; fibrosis;

Chronic pancreatitis stems from relapsing episodes of acute come activated and transition to a myofibroblast-like, alpha- pancreatitis,1 and is characterized by destruction of acinar smooth muscle actin (a-SMA)-positive cell type that is cap- tissue, a sustained pancreatic inflammatory response, and able of depositing large quantities of fibrillar collagen in the fibrosis.2,3 Although development of chronic pancreatitis is interstitial spaces.11,12 The effects of alcohol exposure on PSC strongly associated with heavy alcohol consumption, only a function have been largely deduced from in vitro studies and small percentage of heavy drinkers actually develop the dis- from analysis of human pathological specimens. Studies of ease4–7 suggesting that other predisposing factors (for ex- the mechanisms of alcohol on PSC function in vivo remain ample, genetics, environment, injury) must also be present ambiguous and are confounded by the fact that a good an- for disease progression. Pancreatitis is responsible for ap- imal model of alcoholic chronic pancreatitis (ACP) does not proximately 3500 deaths each year in the USA, 16% of which yet exist.9,13 are due to chronic disease with about half of those deaths due Connective tissue growth factor (CTGF; also known as to alcohol.8 CCN2) is a member of the CCN family of proteins14,15 which Although the pathophysiology of chronic pancreatitis re- associates with components of the extracellular matrix or cell mains poorly understood,9 pancreatic stellate cells (PSC) are surface integrins16 and regulates cellular processes such believed to have a central role in the initiation and progres- as, adhesion, migration, mitogenesis and differentiation.15 sion of the fibrotic response.10 Under normal circumstances Although CTGF has an important role in vertebrate devel- PSC remain quiescent but after pancreatic injury they be- opment17–19 it is weakly expressed in adult connective tissues

1Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA; 2Molecular, Cellular and Program, The Ohio State University, Columbus, OH, USA and 3Department of Surgery, The Ohio State University, Columbus, OH, USA Correspondence: Dr DR Brigstock, PhD, Room WA2022, Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s Hospital, 700 Children’s Drive, Columbus OH 43205, USA. E-mail: [email protected] Received 23 October 2009; revised 18 December 2009; accepted 25 January 2010 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 90 August 2010 1179 CTGF in alcoholic chronic pancreatitis in mice AL Charrier and DR Brigstock

except during wound healing, tissue , or and AEC chromogenic substrate (all from ScyTek Labora- fibrosis.16 A role for CTGF in pancreatic fibrosis was first tories, Logan, UT, USA). Double immunofluorescent detec- proposed from studies that showed CTGF overexpression in tion of a-SMA and CTGF or a-SMA and desmin acute necrotizing pancreatitis20,21 and in desmoplastic re- was achieved by incubation of sections with anti-a-SMA gions of pancreatic cancer.22 In in vitro studies, activated PSC and NH1 anti-CTGF IgY (5 mg/ml; an in-house antibody have been shown to co-express CTGF, transforming growth raised in chickens against recombinant human CTGF) or factor b-1 (TGF-b1), collagen 1 and other extracellular ma- a-SMA and desmin (H:76) (1:50, Santa Cruz Laboratories, trix proteins23,24 and to synthesize CTGF after exposure to Santa Cruz, CA, USA) followed by incubation with ethanol or acetaldehyde.25 However, data showing that CTGF Alexa Fluors 647 goat-anti mouse IgG (1:1000, is expressed by activated PSC in ACP in vivo are lacking. Invitrogen, Carlsbad, CA, USA) and goat-anti chicken Ig- We have produced a rapid and efficient model of ACP in Biotin (1:400, Santa Cruz Laboratories) or Alexa Flour 647 mice that mimics key pathophysiological features of the goat-anti mouse IgG and Alexa Fluor 488 goat-anti rabbit human form of the disease and which shows that activated IgG (1:1000, Invitrogen) for 1 h at room temperature. Slides PSC are a principal source of CTGF in ethanol-induced were incubated with Fluorescent NeutrAvidin (1:200, pancreatic fibrosis. Invitrogen), mounted with Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and examined by confocal laser microscopy (LSM510, Carl Zeiss MATERIALS AND METHODS Co. Ltd, Jena, Germany) using an oil-immersion objective Animal Model lens (Plan-Apochromat  63/NA ¼ 1.4). All animal procedures were approved by the Institutional Animal Care and Use Committee of The Research Institute at Nationwide Children’s Hospital (Columbus, OH). Male In Situ Hybridization C57Bl/6 mice 6–8 weeks old were injected with ethanol Total RNA was extracted from cultured murine PSC with (3.2 g/kg; administered in a 33.3% ethanol: 67.7% water so- RNA STAT-60 (Tel-Test Inc., Friendswood, TX, USA) fol- lution) i.p. one time per day, six times per week, for 3 weeks. lowing the manufacturer’s instructions as previously de- On one day each week, some mice also received an i.p. in- scribed.25 Aliquots of 4 mg of total RNA were reverse jection of cerulein every hour for 6 hours. (50 mg/kg; Sigma transcribed using a SuperScript II Reverse Transcriptase kit Chemical Co., St Louis, MI, USA). Control mice received (Invitrogen). The cDNA was then amplified by polymerase either ethanol alone, cerulein alone, or water alone (n ¼ 6 per chain reaction using primers specific for a-SMA: 50- group). Mice were housed three to a cage and fed a low-fat CTACTGCCGAGCGTGAGATTGTCC-30 (sense) and 50-AGG diet ad libitum. Twice weekly, serum samples were collected GCCCAGCTTCGTCGTATTC-30 (antisense) (497 bp); col- 1 hour after ethanol administration for assessment of peak lagen a(I): 50-GACGTCCCTGGTGAAGTTGGT-30 (sense) blood alcohol levels (BAL). Mice were killed 2 days after the and 50-AGCCACGATGACCCTTTATG-30 (antisense) last ethanol treatment and pancreata were removed before (587 bp); or CTGF: 50-AGTGGAGATGCCAGGAGAAA-30 fixation in 4% paraformaldehyde (pH 7.2–7.4) or for RNA (sense) and 50-ACCTGCACAGCATTTGTTTG-30 (antisense) extraction, as described below. (540 bp). Following initial denaturation at 941C for 2 min, the samples were amplified by 35 cycles of denaturation at Blood Alcohol Levels (BAL) 941C for 30 s, annealing at 601C (621C for CTGF or 651C for Peak BAL were measured using an Ethanol-L3K assay (Di- a-SMA) for 40 s, extension at 721C for 90 s, and ended by agnostic Chemicals Limited, Oxford, CT, USA). NADH extension at 721C for 10 min. The PCR products were se- produced in the assay reaction was quantified by measuring parated on 1.5% agarose gels and then gel extraction was absorption at 340 nm with a SpectraMax M2 Microplate performed using QIAquick Gel Extraction (Qiagen, Valencia, Reader (Molecular Devices, Sunnyvale, CA, USA). CA, USA). Gel extraction products were subcloned into the pCRII-TOPO transcription vector (Invitrogen) according to Histology the manufacturer’s protocol and sequenced using the M13R Fixed pancreatic tissues were embedded in paraffin, cut into sequencing primer. 4 mm sections and stained with haemotoxylin-eosin for Antisense and sense RNA probes labeled with digoxignein- standard histological examination. Collagen staining was UTP or Biotin-UTP were generated by in vitro transcription performed using acidified 0.1% Sirius red F3B (Pfalz & of linearized plasmids with SP6 and T7 RNA polymerases Bauer, Waterbury, CT) as described.26,27 Immunohisto- (Roche Molecular Biochemicals, Mannheim, Germany) chemical staining for a-SMA or F4/80 was performed using according to the manufacturer’s protocol. The digoxigenin- monoclonal mouse-anti human a-SMA (1:100, Dako Cyto- labeled RNA was localized using Anti-digoxignenin-fluor- mation, Denmark) or anti-mouse F4/80 antigen respectively escein, Fab fragments (Roche Molecular Biochemicals). (1:100, eBioscience, San Diego, CA, USA), followed by Biotin-labeled RNA was localized using NeutrAvidin or development with UltraTek Anti-Polyvalent, UltraTek HRP Texas Red (Invitrogen). Finally, slides were washed and

1180 Laboratory Investigation | Volume 90 August 2010 | www.laboratoryinvestigation.org CTGF in alcoholic chronic pancreatitis in mice AL Charrier and DR Brigstock

mounted with Vectashield Mounting Medium with DAPI Statistical Analysis (Vector Laboratories) and examined by confocal laser Values represent mean±s.e.m. of all measurements. Statis- microscopy (LSM510, Carl Zeiss) using an oil-immersion tical analysis of the data was performed using SigmaPlot 11.0. objective lens (Plan-Apochromat  63/NA ¼ 1.4). The Student’s t test was used for paired data that were nor- mally distributed. A P-value of o0.05 was considered sig- nificant. Real-Time PCR Pancreata were removed and immediately immersed in RESULTS RNAlater (Qiagen). RNA was extracted using RNeasy Plus Mouse Model of Alcohol-Induced Pancreatitis Mini Kit (Qiagen) according to the manufacturer’s protocol. As there is limited information regarding the effect of chronic Aliquots of 4 mg of total RNA were reverse transcribed using a administration of intoxicating amounts of ethanol on pan- SuperScript II Reverse Transcriptase kit (Invitrogen). Quan- creatic pathophysiology in mice, we developed a protocol in tification of pancreatic mRNA levels of key marker genes was which C57Bl/6 mice received daily injections of ethanol for 6 achieved by quantitative real-time PCR (ABI PRISM 7000 days per week over a 3-week period. This dosing regimen was Sequence Detection System, Applied Biosystems, Foster City, administered either in normal mice or mice that also received CA, USA) using the following protocol: 951C for 10 min, cerulein treatment once per week to induce recurrent acute followed by 40 cycles of 951C for 15 s and 601C for 1 min, pancreatitis. Within minutes after ethanol injection, all of the then 951C for 15 s followed by a dissociation step of 601C for mice showed physiological signs of intoxication where motor 20 s and 951C for 15 s. Each reaction was run in duplicate and activity, especially of the hind limbs, was highly suppressed all samples were normalized to GAPDH. PCR amplification for at least 4 hours. Over the 3-week dosing period, some of was carried out in a 25 ml reaction containing 0.02 mg cDNA, the mice began to show alcohol tolerance and normal motor 12.5 ml SYBR green PCR Master Mix (Applied Biosystems), activity was resumed within an hour of ethanol administra- 0.16 mM of each primer set. The primer sequences were as tion. In ethanol-treated mice, BALs remained consistently 0 0 follows: CTGF: 5 -CACTCTGCCAGTGGAGTTCA-3 (sense) high at all time points and significantly higher (P 0.05) 0 0 o and 5 -AAGATGTCATTGTCCCCAGG-3 (antisense); TGF- than mice, which received water instead (Figure 1). BALs 0 0 0 b1: 5 -GGTTCATGTCATGGATGGTGC-3 (sense) and 5 -TG were higher during the first two weeks of ethanol treatment 0 0 ACGTCACTGGAGTTGTACGG-3 (antisense); aSMA: 5 -GG as compared with the last week, consistent with acquisition of 0 0 CTCTGGGCTCTGTAAGG-3 (sense) and 5 -CTCTTGC tolerance. In ethanol-treated animals, there was no difference 0 0 TCTGGGCTTCATC-3 (antisense); Collagen a1(I): 5 -GC in peak BALs between mice receiving cerulein as compared 0 0 CCGAACCCCAAGGAAAAGAAGC-3 (sense) and 5 -CTG with those receiving vehicle alone (data not shown). 0 GGAGGCCTCGGTGGACATTAG-3 (antisense); Collagen Livers taken from all groups appeared histologically nor- 0 0 a1(II): 5 -CAAGGCCCCCGAGGTGACAAAG-3 (sense) mal (data not shown) and individual treatments of water/ 0 0 and 5 -GGGGCCAGGGATTCCATTAGAGC-3 (antisense); saline or cerulein alone did not result in pancreatic fibrosis 0 0 Collagen a1(III): 5 -TCCCCTGGCTCAAATGGCTCAC-3 (Figure 2a, b, d, e). Mice that received ethanol alone showed (sense) and 50-GCTCTCCCTTCGCACCGTTCTT-30 (anti- sense); Collagen a1(IV): 50-TCTGCCCTCCTGCGACCTGT-30 (sense) and 50-CGTCTGCCCGCCCTTGTTTC-30 (antisense); TIMP1: 50-TCTGGCATCTGGCATCCTCTTGTT-30 (sense) and 50-CTTCACTGCGGTTCTGGGACTTGT-30 (antisense); TIMP2: 50-GATTCTGCCCCCTCCCCTATTTTC-30 (sense) and 50-GGCCGGCTACACAGTCTTACAACA-30 (antisense); MMP2: 50-CGTCTGCCCGCCCTTGTTTC-30 (sense) and 50- TGCACGGCGATCCCCTTACTCTAC-30 (antisense); a1-in- tegrin: 50-CCGGCCAGGTCGTCATCTACAAG-30 (sense) and 50-CCAGGGGAGCTCCAATCACGAC-30 (antisense); a5-in- tegrin: 50-CTTAGAATCCGAGACTGGGC-30 (sense) and 50-TCAGGACAAATCCCAAACAA-30 (antisense); a6-integrin: 50-ACCGCTGCTGCTCAGAATAT-30 (sense) and 50-AGC- CAGGAGGATGATCCAC-30 (antisense); b1-integrin: 50-AGA AGCTCCGGCCAGAAGACATTA-30(sense) and 50-CCAGC AAAGTGAAACCCAGCATCC-30 (antisense); DDR2: 50-GTGT Figure 1 Peak blood alcohol levels. BALs were assessed within 1 h of GGGCCTTTGGGGTGACTCT-30 (sense) and 50-ATGGCCG ethanol administration. During the first 2 weeks of ethanol administration, 0 0 mean peak BALs remained above 400 mg/dl and dropped during the last GTGCTTGGTTTCTCTTC-3 (antisense); and GAPDH: 5 - week of treatment to 300–400 mg/dl. Peak BALs in mice receiving water 0 0 TGCACCACCAACTGCTTAGC-3 (sense) and 5 -GGCATGG remained consistently low throughout the duration of the experiment. 0 ACTGTGGTCATGAG-3 (antisense). *Po0.05, **Po0.001. www.laboratoryinvestigation.org | Laboratory Investigation | Volume 90 August 2010 1181 CTGF in alcoholic chronic pancreatitis in mice AL Charrier and DR Brigstock

1182 Laboratory Investigation | Volume 90 August 2010 | www.laboratoryinvestigation.org CTGF in alcoholic chronic pancreatitis in mice AL Charrier and DR Brigstock

some pancreatic fibrosis, but this was typically confined to hybridization was undertaken to assess mRNA localization isolated areas of peripheral fat tissue (Figure 2g, h, i, n). On for collagen a1(I) or a-SMA. When the same tissue sections the other hand, mice that received both ethanol plus cerulein were co-stained for each molecule and assessed by confocal showed many features of chronic pancreatitis that included microscopy, the merged fluorescent images clearly showed loss of parenchymal structure and extensive areas of par- that collagen a1(I) mRNA and a-SMA mRNA localized to the enchymal fibrosis, which was both perilobular and in- same cell (Figure 5b). tralobular in nature (Figure 2j, k) and also apparent in Next, since CTGF is strongly implicated in pancreatic peripheral fat (Figure 2p, q). Although acinar damage and fibrosis and has been shown to be produced in activated PSC collagen deposition were very evident, these features did not in vitro,10 we further analyzed the sections for the presence of occur uniformly throughout each tissue section, with some CTGF protein or mRNA. As assessed by immunofluorescent areas retaining their normal structure. staining with anti-CTGF IgG, CTGF was present in all a-SMA immunoreactivity was restricted to the smooth treatment groups within the vasculature (data not shown) as muscle vasculature in mice that received water/saline or previously documented in several other normal tissues and cerulein alone (Figure 2c, f). In contrast, extensive a-SMA organs.28 However, CTGF protein was additionally localized staining of myofibroblastic cells (that is, presumptive acti- to the collagen-rich areas adjacent to necrotic parenchymal vated PSCs) was seen in parenchymal tissue surrounding tissue in mice that received ethanol plus cerulein (Figure 6a) areas of necrosis of animals receiving both ethanol plus cer- as well as to the peripheral fat in these mice (data not shown) ulein together (Figure 2l). These infiltrating myofibroblasts or in mice receiving ethanol alone (Figure 6e). When tissue were also present in the peripheral fat tissue of mice that sections from fibrotic mice were further analyzed by double received either ethanol alone or ethanol plus cerulein (Figure staining with either CTGF and a-SMA antibodies or CTGF 2o, r). As shown in Figure 2k-l, accumulation of a-SMA- and a-SMA mRNA probes, the merged confocal images positive cells was closely associated with areas of collagen showed that both molecules were localized to the same cells deposition. The increase in a-SMA-positive cells or collagen deposition in treated animals was further quantified by image analysis of the stained slides. The area of positive collagen deposition or a-SMA-positive cells was significantly increased (430%; Po0.001) in animals receiving ethanol plus cerulein as compared with all other treatment groups. The increase in a-SMA-positive cells was only significant (Po0.05) in ani- mals receiving ethanol alone as compared with either cerulein alone or water/saline groups (Figure 3). Animals receiving water/saline, cerulein alone or ethanol alone did not show activated macrophage infiltration as shown by F4/80 immunohistochemical staining (Figure 4a-c), whereas animals receiving both ethanol and cerulein showed persistent infiltration of mononuclear cells (activated macro- phages) in areas of fibrosis and necrosis (Figure 4d). Taken together, these data show that the mice, which re- ceived ethanol plus cerulein showed typical features of hu- man ACP including parenchymal loss, sustained Figure 3 Image analysis of collagen or a-SMA staining. Quantification was inflammatory response, myofibroblast accumulation and performed by image analysis of 30 representative images ( Â 200) from extensive fibrosis. three animals per treatment group by ImagePro Plus software. Collagen and a-SMA staining were increased significantly in pancreata from animals treated with ethanol plus cerulein as compared with pancreata from all Co-Production of Collagen and CTGF By Activated PSC other treatment groups. Staining differences were not statistically To verify that presumptive activated PSC were a principal significant for either water þ cerulein vs water þ saline or ethnaol þ source of collagen in the mouse ACP model, in situ saline vs water þ saline. *Po0.05, **Po0.001.

Figure 2 Histological changes in mouse pancreatic tissues after chronic administration of ethanol. Mice (n ¼ 6 per group) received water/saline (a–c), cerulein alone (d–f), ethanol alone (g–i, m-o) or ethanol plus cerulein (j–l, p–r) for 3 weeks as described in Materials and Methods. Pancreatic tissue sections were subsequently stained with H&E (a, d, g, j, m, p), Sirius red (b, e, h, k, n, q) or anti- a-SMA (c, f, i, l, o, r). Pancreata from mice that were treated with water/saline (a–c), cerulein alone (d–f) showed no overt pathology, with no evidence of increased collagen deposition or increased a-SMA production. In these mice, collagen and a-SMA were restricted to, respectively, the supporting structure and smooth muscle lining of the vasculature. Mice treated with ethanol alone showed no overt pathology other than minor collagen deposition and aSMA staining in focal areas of peripheral fat tissue (m–o), which was also seen in mice treated with ethanol plus cerulein (p–r). In contrast, pancreata of mice treated with ethanol plus cerulein (j–l) showed massive collagen deposition and a highly correlated and intense amount of a-SMA staining in and around areas of acinar degeneration and necrosis. ( Â 100: a–l, Â 400: m–r).

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Figure 4 Macrophage infiltration in diseased animals. Mice (n ¼ 6 per group) received water/saline (a), cerulein alone (b), ethanol alone (c) or ethanol plus cerulein (d) for 3 weeks as described in Materials and methods. Pancreatic tissue sections were subsequently stained with anti-F4/80 to detect the presence of activated macrophages. Positive staining was only detected in animals receiving ethanol plus cerulein (d) and was localized to areas of fibrosis within perilobular fat tissue and the intralobular spaces ( Â 400).

at the protein (Figure 6a) and mRNA (Figure 5a) level. of these key markers in alcoholic pancreatic fibrosis was Normal parenchymal tissue in either control mice or from characteristic of activated PSC. the unaffected regions in mice receiving ethanol or ethanol plus cerulein failed to show any staining for CTGF (Figure Changes in mRNA Expression of Fibrotic Markers in ACP 6d-h). As desmin is produced by both activated and queis- To verify that ACP in mice resulted in the expected changes cent PSC,10 it was of interest to determine whether the in mRNA expression of key profibrotic molecules, quantita- a-SMA-positive cells in fibrotic regions were also desmin- tive real-time PCR was performed on total pancreatic RNA positive. Double immunofluorescent staining for a-SMA and isolated from animals that received either water plus saline or desmin showed that these proteins did indeed co-localize to ethanol plus cerulein. In support of the data shown in Figures the same cells (Figure 6c) although high power analysis of the 2–6, expression of mRNA for CTGF, a-SMA, or collagen merged images revealed that the signals within each cell did a1(I) was significantly increased (Figure 7). Similarly, the not overlap with one another (Figure 6c, lower right) con- expression of TGF-b1, collagen a1(II), collagen a1(III), tissue sistent with their different intracellular distribution. Collec- inhibitors of metalloproteases-I and –II, discoidin domain tively, the data revealed an intimate relationship between receptor 2, and the integrin a1, a5, a6, and b1 subunits in collagen a1(I), a-SMA, desmin, and CTGF in that expression fibrotic mice was also significantly increased relative to

1184 Laboratory Investigation | Volume 90 August 2010 | www.laboratoryinvestigation.org CTGF in alcoholic chronic pancreatitis in mice AL Charrier and DR Brigstock

Figure 5 Co-localization of CTGF mRNA and collagen a1(I) mRNA to a-SMA mRNA-positive cells. In animals receiving both ethanol plus cerulein, confocal microscopy showed that CTGF (a) and collagen a1(I) (b) mRNA were co-localized to areas of a-SMA mRNA (that is, presumptive activated PSC) in areas of intense fibrosis within the pancreatic parenchyma . Co-localization of CTGF, collagen a1(I) and a-SMA mRNA was also observed in the fibrotic regions in peripheral fat (see Figure 2m-r) in mice receiving either ethanol plus cerulein or ethanol alone (data not shown). ( Â 630). control mice (Po0.05) (Figure 7). Expression of collagen a1 isms of this disease. Several models of chronic pancreatitis in (IV) or matrix metalloprotease-II was not significantly mice rely on administration of cerulein alone, but there is changed (Figure 7). uncertainty as to the relevance of these models as it relates to alcohol-mediated injury. The development of a mouse model DISCUSSION is particularly difficult because mice metabolize ethanol at a Both acute and chronic pancreatitis are associated with long- rate of up to 550 mg/kg/h as opposed to 100 mg/kg/h in term heavy alcohol consumption. Acute pancreatitis is humans32 and, like humans, mice rarely develop fibrosis characterized by pain, edema, hemorrhage, acinar cell upon ethanol administration alone.33 Peak BALs may also be vacuolation, necrosis, and increased serum amylase and a more important factor in assessing potential tissue damage lipase; these events are confounded in the chronic form of the than actual ethanol dose administered because of varying disease by fibrosis, inflammation, calcification, and decreased metabolic differences34,35 and mice are considered to be exocrine and endocrine function. One theory for the devel- heavily intoxicated when peak BALs are 400–500 mg/dl.36 opment of chronic pancreatitis is the necrosis–fibrosis hy- While intragastric ethanol infusion of mice is able to achieve pothesis,1 which proposes that the disease originates from high BALs,36 this approach is costly, labor-dependent, time repeated tissue injury due to recurrent episodes of acute consuming, and not readily amenable to most researchers. pancreatitis. This has been further developed to also re- Ethanol-containing diets have also been employed in mice cognize a potential sentinel acute pancreatitis event,29 which and rats but these too are labor-dependent, time consum- initiates activation of the immune system, mainly in the form ing33,37,38 and are particularly problematic because heavy of activated myofibroblasts (including PSC) and infiltrating intoxication is rarely achieved.13 mononuclear cells. Through their release and action of cy- In this study, we developed a new model of ACP in mice to tokines, chemokines and other stimulatory factors, PSC are complement our previous studies of the role of CTGF in proposed to exist in a persistently activated form10 and to pancreatitis21 and PSC biology.25,39,40 Important features of promote fibrosis30,31 through their unrelenting production this model were (i) ethanol administration; (ii) achievement and deposition of collagen. of high, intoxicating peak BAL; (iii) a background of Whereas chronic pancreatitis in humans is frequently due recurrent acute pancreatitis against which the effects of to excessive alcohol consumption, this etiology has proven ethanol could be evaluated; and (iv) pathophysiology consi- challenging to study in mice, even though the diverse genetic stent with human ACP. The data reported herein show that backgrounds offered by this species would potentially allow all of these criteria were met in the mouse model described important insight into the underlying pathological mechan- which was also reproducible, time- and cost-efficient. The

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Figure 6 Immunohistochemical co-localization of CTGF, a-SMA, and desmin to presumptive activated PSC. Pancreata from mice receiving ethanol plus cerulein (a–d), ethanol alone (e–f), cerulein alone (g) or water/saline (h) were processed by immunofluoresent staining to detect CTGF, a-SMA, or desmin. Confocal microscopy of animals receiving ethanol plus cerulein showed co-localization of CTGF and a-SMA in presumptive activated PSC in areas of fibrosis adjacent to areas of necrosis (a–b). Cells positive for a-SMA were also positive for desmin (c), although both proteins were localized to different regions of the same cell, as expected. In non-fibrotic areas of the pancreata from these mice, CTGF and a-SMA expression were only detected in blood vessels (d). In animals receiving ethanol alone, CTGF and a-SMA were expressed only in areas of peripheral fat tissue (e) whereas pancreatic parenchymal tissue did not express CTGF or a-SMA (f). (a, d–h)  630; (b)  630 with  3 zoom; (c)  630 with  4 zoom, except bottom right picture which is  3.5 magnification of original merged panel. overall strategy was to adopt a ‘dual-hit’ approach in which required to be intoxicating in this otherwise highly resistant acute episodes of cerulein-induced pancreatitis sensitized the species. Careful analysis of the pancreata obtained from mice pancreas to severe damage by chronic ethanol exposure. treated in this manner, as compared with mice receiving Moreover, administration of the extremely high doses of control treatments such as alcohol alone or cerulein alone, ethanol resulted in peak BALs that were of the magnitude showed a highly reproducible pathogenesis that was typified

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intralobular and perilobular fibrosis, sustained inflammation, necrosis, and parenchymal loss—are readily manifested. The short time frame of our protocol substantially reduces animal housing costs and allows for more efficient data collection and analysis. Finally, when animals are maintained on the Lieber-DeCarli diet, feeding must be strictly monitored for caloric intake, specialized feeding equipment is required, each animal must be housed singly, and water intake is not allowed,9 whereas our model offers the advantages that there is no need for ingestion of an isocaloric diet, ethanol intake can be exquisitely controlled by simple i.p. injection, mice can be maintained three to a cage on normal low-fat diets, where each animal is able to drink water and feed ad libitum,and animal husbandry is overall more efficient and cost-effective. The damaging effects of alcohol on the pancreas are sig- nificant and multi-faceted, at least in part because ethanol is Figure 7 Pancreatic expression of fibrosis-related genes. Quantitative real- locally metabolized, generating toxic metabolites that cause time PCR was performed on total pancreatic RNA isolated from mice that or exacerbate pancreatic injury. Although pancreatic acinar received either ethanol plus cerulein or water/saline (n ¼ 6 per group). *Po0.05 l. cells have been well characterized in terms of their ability to metabolize ethanol via either oxidative and non-oxidative pathways,41 data are also available which support a role by persistent mononuclear cell (activated macrophages) for PSC in either metabolizing ethanol or functionally infiltration, parenchymal loss, necrosis, accumulation of responding to ethanol metabolites. For example, we have a-SMA-positive myofibroblasts (that is, presumptive acti- shown that ethanol or acetaldehyde stimulate CTGF pro- vated PSC), and fibrosis. moter activity and CTGF protein production in rat PSC39 Histochemical findings confirmed that aSMA-positive cells and that the production of acetaldehyde and oxidant stress in were located in fibrotic areas adjacent to regions of acinar mouse PSC are the cause of increased CTGF mRNA and necrosis and that the same cells were positive for the production protein production after exposure of the cells to ethanol.25 In of desmin, collagen, and CTGF. These cells, which presumably view of our findings that PSC are the principal producers of comprise the activated PSC population (although resident CTGF in murine ACP (these studies) and that PSC treated myofibroblasts cannot be excluded), thus appear to be the with recombinant CTGF show increased adhesion, migra- principal pro-fibrogenic cell types in murine ACP in as much as tion, proliferation, DNA synthesis, and collagen I synthesis,40 they were the main producers of both CTGF and collagen. This additional in vitro and in vivo studies regarding the role of finding is consistent with studies of human ACP.20 alcohol in PSC-mediated CTGF production will likely yield In a previous study, C3H mice developed pancreatic important clues as to disease pathogenesis and possible fibrosis after receiving the Lieber-DeCarli ethanol diet for therapeutic strategies. 8 weeks coupled with i.p cerulein injections delivered on Despite a clear role for CTGF in the regulation of PSC alternating days for the last 1–5 weeks.33 It is notable that this function and its production by activated PSC both in vitro and regimen is not applicable to the C57Bl/6 mice used in our in fibrosing pancreatic tissues in vivo, additional studies study because, even in the absence of ethanol, administration addressing its precise role in pancreatic fibrosis are required in of cerulein three times a week to C57Bl/6 mice results in light of emerging data from CTGF transgenic mice that have profound pancreatic fibrosis (data not shown). In fact the highlighted the importance of concurrent injury cascades in authors of the earlier study33 reported that in C3H mice organs such as the liver or for the manifestation of cerulein alone caused PSC activation and increased expres- CTGF fibrotic activity.42–44 Collectively these studies have sion of TGF-b1 and TIMP-1 which, although transient in shown that CTGF likely exerts a fibrotic action by acting as a nature, may confound the study of ethanol-specific effects in portal or hub through which cells are able to integrate multiple some studies. It is also important to recognize that genetic molecular cues and to respond with a pro-fibrotic output. background strongly influences susceptibility to develop Understanding of this phenomenon in pancreatic tissues is pancreatic fibrosis and, accordingly, the development and thus a prerequisite for elucidation of the mechanism of action validation of more than one mouse model of ACP is a of CTGF in driving pancreatic fibrosis. Nonetheless, the data definite advantage given the numerous mouse strains that are presented in this study show that collagen-producing a-SMA- available to study. Nonetheless, our model offers several positive PSC act as a central player in CTGF production in advantages including the fact that severe ethanol-mediated ACP in vivo and highlight the importance of activated PSC pancreatic pathology develops within a short time frame and CTGF in the pathophysiological mechanisms involved as (3 weeks) and that key features of ACP—profound well as their potential as targets for anti-fibrotic therapies.

www.laboratoryinvestigation.org | Laboratory Investigation | Volume 90 August 2010 1187 CTGF in alcoholic chronic pancreatitis in mice AL Charrier and DR Brigstock

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