Acid-pepsin injury to rabbit esophageal epithelium

blot arrays of >18,000 murine EST sequences (Mouse GDA, Incyte Genomics). Differential expression of selected ESTs was confirmed by RNase protection assay using riboprebes prepared from sequenced linearized plasmids from which the ESTs were derived. Relative expression levels of pancreatic transcripts were compared to the pancreatic housekeeping transcript of murine acidic ribosomal protein PO (mARP). RESULTS:A sequence identified as mouse prostatic secretory glycoprotein (MPSG), a serine proteaseinhibitor, was found to be overexpressed2.3-fold in the array analysis. The plasmid containing this EST (Incyte Genomics) was sequenced and the insert was established to be a full-length sequence of MPSG, now termed SPINK3. Following a single 6 hr treatment with cerulein, 50/~g/kg/hr, SPINK3 mRNA increased2 to 2.5-fold at 24-72 hr over the relativelyabundantbasaltranscript levels. The induction of SPINK3 was dose-dependent:no induction followed physiologic or sub-injurious cerulein stimulation (0.1 to 5/~g/kg/hr) whereas 10-50/~g/kg/hr induced a 1.8 to 2.6-fold increase in SPINK3 mRNA levels at 24 hr. CONCLUSION:These experiments identified pancreaticSPINK3 as a gene induced following acute or repetitive pancreaticinjury in the mouse. Genetic polymorphisms of a similar serine protease inhibitor, SPINK1, may confer an increased risk of chronic pancreatitis (PfOtzer, Gastro 119:615, 2000). Therefore, pancreaticSPINK3,which we show hereto have abundantbasaltranscript levels in the mouse and to be induced by injury, could be another protease inhibitor important in preventing repetitive acinar cell injury by prematurely activated digestive enzymes.

598 Matrix Metalloproteinase-9 (MMP-9) Enhances Leukocyte Trafficking and Alveolar Capillary Leakage in Pancreatitis-Associated Lung Injury by Basement Membrane Digestion Tobias Keck, James H. Balcom IV, BozenaA. Antoniu, Carlos Fernandez-Del-castillo, Andrew L. Warshaw, Dept of Surg, MA Gen Hosp, Boston, MA Background: In pancreatitis-associatedlung injury invading neutrophils (PMN) access the lung by migration through capillary endothelial basement membranes. We hypothesizethat this process may be facilitated by digestion and degenerationof the basement membrane by specific PMN-producedmatrix metalloproteinases(MMPs). Methods: Mild or severepancreatitis was induced in rats. MMP-2 and MMP-9 activity in supernatant of PMN cultures, and homogenatesof lungs were assessed by zymographyand Western blot. Congruenceof PMN and MMP expressionin lung tissue was evaluatedby confooalmicroscopy (CM) andfluorescent immunohisto-chemistry (IHC) for MMP-9 and neutrophils. The contribution of MMPs to PMN transmigration was tested with the MMP-inhibitor batimastat (BB-94) in vitro (TNF-~-induced PMN transmigration across matrigel chambers) and in vivo (myeloperoxidaseactivity and Evans blue concentration in broncho-alveolar lavage fluid - BALF). Results: MMP-2 (72kD gelatinase) was constitutively expressed in both, controls and animals with pancreatitis. By contrast MMP-9 (92kD gelatinase)was highly expressedin lungs and supernatantof neutrophil cultures only in severe pancreatitis. IHC and CM showed colocalisation of MMP-9 and PMN in lung tissue. BB-94 inhibition of MMPs in vitro significantly reduced TNF-~induced PMN transmigration across matrigel (10.85_+.61 vs. 2.75__..29cells per visual field, p<0.001). In severepancreatitislung injury was significantly amelioratedby BB-94, as indicatedby reduced myeloperoxidaseactivity (39.7_+.5 vs. 22.9-+.8 mU/mg protein, p<0.05) and alveolarcapillary leakage (Evans blue in BALF 11.48-+.28 vs. 4.39-+.09 % of serum Evans blue, p<0.05). Conclusion: Our data indicate that MMP-9 secretion by PMN increases in pancrcatitis in proportion to the severity of pancreatitis.MMP-inhibition reduces PMN transmigration in vitro and in vivo and reduces resultant alveolar-capillaryleakage.Thesefindings suggest an important role for MMP-9 in the pathogenesisof pancreatitis - associated lung injury.

6OO Peroxisome Proliferator-Activated Receptor (PPAR)-,y Ligand Inhibits The Progression Of Pancreatic Fibrosis In Vivo And Prefibrogenic Aclion In Pancreatic Myofibroblast. Kyoko Shimizu, Shoko Hicada, Keiko Shiratori, Makio Kohayashi,Naoaki Hayashi,Tokyo Women's Medical Univ, Tokyo Japan Background: We have reported that ligand of nuclear hormone receptor superfamily of transcription factor PPAR-~improved pancreaticinflammatory responsesmediatedvia the modulation of proinflammatory cytokine production and NF-KB activation on activated macrophages in animal model of chronic pancrsatitis. Herewe show that PPAR~ ligand, troglitazoneinhibits the pancreatic fibrogensis in vivo and in vitro. Methods: I) In vivo study: 0.2% troglitazone containing rats chow was administered from one month to seven months of age in WBN/ Koh rats with spontaneouschronic pancrsatitis.The areaof fibrotic tissue stained by Masson's tdchrome was calculated using an image analysis. Expressionof collagen I, III, fibronectin, and ~-smooth muscle astin (e-SMA) was evaluated by immunohistochemical study and Western blot analysis. Pancreatichydroxyproline levels were measured by HPLC. 2) In vitro study: Pancreaticstellate cells freshly isolatedfrom rat pancreaswere cultured and characterized as pancreatic myofihrohlasts morphologically. Expressionof PPAR--/and ~-SMA were determined by immunohistochemical studies and Western blot analysis. Cell proliferation or cell cycle was analyzedby MI-I assay or flow cytometory. Results: 1) Area of fibrotic tissue in troglitazone-treatedrats was significantly less than in control ra~ (4-months old rats: 5.6% vs 1.2%, 7-months old rats: 4.2% vs 3.6%, respectively). Expression of collagen I, III, fibronectin, and e-SMA were also significantly reduced by the administration of troglitazone. Tissue levels of hydmxyproline in 4-months (8.3/.cmol/g) and 7-months old (7.4/d~ol/g) tmglitazone-treated rats were lower than in control group (12.0 /.Lmol/g or 11.3 /.~mol/g, respectively). 2) Pancreatic myofibroblasts expressed PPAR-~ in the nucleus and ~-SMA in the cytoplasm from 2-6 passages. Cell proliferation stimulated by platelet-dedved growth factor (PDGF) was inhibited by tmglitazone in a dose-dependentmanner (0.001-10/~M). Percentageof cells stimulated by PDGFwas increased in the S phase and decreasedin the G1 phase.Addition of troglitazone suppressedthe rise of percentageof cells in the S phase. Conclusion: PPAR-~ ligand inhibited the progression of pancreaticfibrogenesis, mediatedvia the modulation of proliferation in pancreatic myofihroblasts.

601 Hypoxia Regulates Angioievasion and Metastasis of Pancreatic Cancer by Activaiton of HIF-1, a New Regulator or uPAR Expression. Peter Buechler, Chirurgie I, UIm Germany; Joe O. Hines, Manuela C. Buechier, James S. Tomlinson, UCLA Sch of Medicine, Los Angeles, CA; Helmut Friess, Markus W. Buechler, Clin fuer Visceralchirurgie, Bern Switzerland; Hans G. Beger, Chirurgie I, UIm Germany; Howard A. Reber, UCLA Sch of Medicine, Los Angeles, CA

599 Acute Pancreatitis and Systemic Inflammatory Response Induced by NF-KB Activation in Pancreas Xueqing Chen, Baoan Ji, Bing Han, Stephen A. Ernst, Diane M. Simeone, Craig D. Logsdon, Univ of Michigan, Ann Arbor, MI Background: The cellular mechanisms that initiate acute pancreatitis are uncertain. While premature activation of the digestive enzyme trypsinogen is widely believed to be the key event, the transcription factor NF-KB is also activated early in acute pancreatitis. NF-KB is considered a key element in inflammatory responses based upon its ability to regulate the expression of inflammatory mediators in vitro, though its role in vivo in disease remains unclear. Methods: In the current study we directly activated NF-KB within the pancreasusing adenoviral mediated transfer of an active subunit, RelA/p65 (Adp65). This adenovirus was deliveredby intraductuleinjection. Viral deliverywas verified and quantitatedusing the expression of GFP that occurred from a separatepromoter in Adp65. Activation of NF-KB signaling was verified by monitoring the expression of the NF-KB responsivechemokine gene, mob1.Animals injectedwith Adp65 or a control adenoviruswere analyzedfor parametersassociated with pancreatitis including levels of serum amylase and tissue water and myelo-peroxidase (MPO) activity in pancreasand lungs. Thesetissues were also examinedhistologically. Leve~s of trypsinogen activation were monitored using an in-gel trypsin assay. Results: Intraductule delivery of Adp65 led to infection of a minority (-8%) of pancreatic acinar cells, increased levels of RelNp65 and expressionof an NF-KB responsivegene. Injection of Adp65 increased pancreatic edema, MPO activity, and neutrophilic infiltration and causedwide-spreaddamage to pancreatic acinar cells= increases in lung MPO activity and neutrophilic infiltration were also observed. In contrast, control adenovirushad only minor and transient effects on pancreatic edema and not other parameters. Trypsinogen activation did not play an important role in the response to Adp65 as trypsin activity was increasedto a minor and equal extent in animals administered either Adp65 or control adenovirus. Conclusions: Activation of NF-KB within the pancreas is sufficient for the initiation of acute pancreatitis. These results help define the specific role of NF-KB activation in a complex inflammatory disease.

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Background: Angioinvasion is the rate limiting step in metastatictumor progression. Among the very few proteins ultimately necessaryfor angioinvasion is urokinase receptor (uPAR). Hypoxia-lnducible-Factor-1(HIF-1) is found to be activatedduring growth of human pancreatic cancer, a tumor which grows in a microenvironment of low oxygen. Transcriptional control of uPAR expression by HIF-1 has never been reported. Aim: The aims of this study were to analyzewhether HIF-1 transcriptionally regulates its potentially new target gene uPARand to analyzethe role of hypoxia in angioinvasion and therefore metastasis Methods: Four human pancreatic cancer cell lines Capan-2 (C2), HPAF-2 (HP2), MIA PaCa-2 (MP2) and PANC-1 were used. Northem blot analysis was used to quantify mRNA expression. Nuclear run off assaysto determine newly transcribed mRNA. DNA binding was studied with Gelshift assays. PCR generated deletion mutants of the uPAR promotor were used in Luciferase assays to determine reporter gene activity. Matrigel invasion assays and fertilized chicken eggs were used for chodoallantoic membrane (CAM) assay to study angioinvasion of PaCacell lines in vitro and in vivo and PCR to specifically detect human sequences in the opposite CAM. Results: uPAR but not uPA mRNA expression is upregulatad by low oxygen levels. Cell lines not expressing uPAR under normoxic (C2) conditions reconstitute uPAR production under hypoxia. Actual hypoxia induced uPAR mRNA transcription was found up to 7.2_+1.6 fold increased after a 12h exposure. Transcriptional activation of HIF-f by binding to the uPAR promotor was detectable within the first hour after onset of hypoxia. Promotor deletion mutants containing the putative HIF-1 binding site showed up to a 164_+5.5 fold increase in reporter gene activity. In vitro invasion assays showed that hypoxia increased the relative number of invading tumor cells (0.22_+0.07 vs 0.09-+0.02). Moreover, hypoxia was a strong uPAR dependentstimulus in angioinvasionassays in vivo in all cell lines tested. Conclusion: In this study we present for the first time, that uPAR is under transcriptional control of HIF1 and further show that hypoxic upregulation of uPAR is caused by an actual increase in transcription of uPAR mRNA synthesis. We further show that this mechanism also increased the potential of metastaticgrowth of pancreatic cancer, both in vitro and in vivo in an uPAR dependent manner. This study was supported by the R.S. Hirshherg Foundation

602 Acid-Pepsin Injury To Rabbit Esophageal Epithelium Nelia A. Tobey, Seraj S. Hosseini, Roy C. Orlando, Tulane Univ Sch of Medicine and VA Medical Ctr, New Orleans, LA BACKGROUND:A componentof the refluxateof humans is pepsin,an acid-activatedprotease, secreted by gastric chief cells. Pepsinat acidic pH has beenshown to damagethe esophageal epithelium (EE) to significantly greater degrees than the same acidity alone. However there is little data on the nature of the early acid-peptic lesion, its potential reversibility or the mechanism by which this pepsin increasesthe rate of acid damageto EE. METHODS:In the present study this information was sought by exposing Ussing-chamberedsections of rabbit EEand cultured rabbit EEcells to acid-pepsinat varying concentrationsand pH. In the Ussing chamber, damage by acid-pepsin was assessed by decline in electrical resistance (R), light and transmission electron microscopy (TEM) and by mannitol flux; while damageto cultured cells was monitored by trypan blue (T.B.) uptake. RESULTS:(Means_+SEM,n>3). HCI, pH

1.7, reduced R of rabbit EE in lh by 43_+5% while exposureto acid-pepsin, 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml reduced R by 61_+3%, 86_+3% and 89_+5%, respectively.After lh at pHs of 1.7, 2.0, 2.25, 2.5, and 3.0, acid-pepsin, 1.0 mg/ml, reduced R by 99_+0.1%, 95-+3%, 94_+2%, 83_+5%, and 16_+5% respectively. Exposure to acid alone, pH 1.7, for 15 min decreasedR by 18_+5%and on neutralizationR returnedto baselinecomparedto acid-pepsin, pH 1.7 + pepsin, 1 mg/ml, which reduced R by 52_+15% with little to no recovery after neutralization. Moreover, the decline in R with acid-pepsin was associated with an increase in mannitol flux in the absence of change on light microscopy. For primary cultured EE cells growing on glass coverslips, the % T.B. uptake was greater with acid-pepeinthan acid alone at pH 2 and 1 but not pH 3; but this difference in % T.B. uptake was abolished if EE cells were exposedto acid or acid plus pepsinwhile in suspension.CONCLUSIONS:Pepsindamages the EE at concentrations >0.15 mg/ml and at acidic pH values of < 3. Pepsin enhancesthe rate and degree of injury to EE at acid pH by irreversibly damagingthe intercellularjunctional complexso that it increasesthe rate of acid accessto the cell's more acid permeablebasolateral membranes. Support:NIHDK-36013. 603 Transient Lower Esophageal Sphinder Reluxalioux Are Nor the Result of Mechanical Opening of the Cardla Doting Gastric DIMemdon Benson T. Massey, Chelsey Simuncak, Nicole J. Lecapitaine-Oana,Sudhindra Pudor, Medical Coil of Wisconsin, Milwaukee, WI BACKGROUND: Transient lower esophageal sphincter relaxations (TLESRs) are the main mechanism for gastroesophagealreflux events. Several studies have provided evidencethat these are neurally mediated events involving the vagus nerve. However, another school of thought is that these manometrically-observedrelaxations are in fact the result of gastric distension, which distracts the cardiato open the lumen and thus reduceintraluminal pressure. Studies in support of this hypothesis have used in vitro human or in vivo a n e s t ~ animal models. AIMS: To assess the relationship of manometric TLESR to endoscopic opening of the cardia in an awake human model. METHODS: 7 healthy volunteers (6M/1F, age 18-53) with no endoscopic evidence of hiatal hernia were studied in a sitting position. Manomeby was performed using a sleeve catheter passed through 1 nostril. Through the other nostril, a 5.3mm fiberoptic endoscopewith a videoadaptorwas passed and positioned so as to have a retroflexed view of the cardia. The 2ram channel of the endoscope was connected to a barostat to allow distension of the stomach at a target pressure of 15mmHg. Distension sessionswere performedfor 30 minutes, with a concurrent timing signal sent to the manometric tracing and the videorecorder. TLESRs were identified by standard criteria, and the time of onset, nadir, and end were recorded. Times of first opening and final closure of the gastroesophagealjunction (GEJ) were recorded independentlyof the manometric findings. RESULTS: 142 TLESR events were available for analysis. Onset time of TLESRs invariably precededthe first sign of GEJ opening [6.3s (range 0.5-20.7s, p<=O01]. Although the TLESR nadir often closely approximated the time of GEJ opening, the nadir could occur several seconds before the GEJ was seen to suddenly snap open, at which time common cavity pressures were typically seen on manometry. Once open, the GEJ moved proJdmallyfor the duration of the TLESR.Termination of TLESRs by primary or secondaryperistalsis occurred about the time the time of GEJ closure (net difference 0.1s, p = NS). Closuretended to occur before the cardia completed its aborad move to the rest position. HenceTLESR events ware longer than GEJ openingtimes (17.0 vs. 12.6s, p<.001). CONCLUSIONS:Thereis no evidence for TLESRs being the passive result of mechanical cardia distraction. Rather, TLESRs must occur first before the cardia can open. Supported by P01 03191-02.

NL GERD FP

OP (mmHg) 24.8±1.3 16.0±1.2" 21.7±1.8

Plcm (mmH9) 16 9±2.5 7.2_+0.9* 15.4+1.2

Diameter(cm) 15 mmH9 25 mmH9 1.1±0.1 1.5±0.1 1 5:L~).1* 2.0±0.1" 1.2_+0.1 1.5:L-0.1

HC Length (cm) 1.3-/-0.1 1.0-1-0.1" 2.0~.1#

• p < 0.05vs NL & vs FP #p < 0.05vs NL Ptcm: pressurewith diameter> 1cm. HC: Hitalcanal. OP: openingpressure. 605 Do Patients With Reflux Disease Have More 6antroesophageal Reflux Episodes 1loan Noaltby SabJesls? Acid And Non-Acid Reflux Dudng 24-Hr Ambulatoiy pHImpeflaM P . m ~ z e s Daniel Sifrim, K U Leuven, Leuven Belgium; Richard H. Holloway, Royal Adelaide Hosp, Adelaide Australia; Jiri Silny, Helmhoifz Institute, Aachen Germany; Jan Tack, Jozef Janssans, K U Leuven, Leuven Belgium It is generallyacceptedthat patients with GERDhave more acid reflux episodesthan healthy subjects, suggesting that failure of the antireflux barrier is critical in the pethogenesis of GERD.This concept derives from measurementsof esophagealpl-I, where reflux was defined as a fall in pH below 4.0. However,the refluxate might be less acid or non-acid depending on the available fuodic contents. The rate of reflux events is relevant when evaluating the effect of therapies directed at improving the antireflux barrier function. We aimed to measure the total rate of reflux episodes (acid and non-acid) in patients with GERD in ambulatory conditions. Methods: 24-hr ambulatory pH-impedance recordings were performed in 30 patients with erosive esophagifis and 28 controls. Tracings were visually analyzedand reflux was classified as traditional acid (pH drop below 4.0), minor acid (pH drop above 4.0) or non-acid (pH drop less than 1 unit + liquid reflux in impedance)and the refluxate could be pure liquid, or mixed of liquid and gas. Results: Gastroesophagealreflux of gastric contents was equally frequent in both groups. However, patients with GERD had a higher rate and proportion of traditional acid reflux (table). The rate of minor acid and non-acid reflux was similar in patients and controls. One third of reflux events was non-asid. Mixed reflux of gas and liquid was the most frequent pattern with gas preceding liquid in 50-80% of cases. Pure liquid reflux was more often traditional acid in patients with GERDthan in controls (45% vs. 32%, p < 0.05). The puttems of reflux were not influenced either by the severity of GERD or the presenceof hiatus hernia.Conclusions: Refluxof gastric contents was similarly frequent in patients with GERDand controls. However,patients with GERDhave more acidic refluxatss suggesting either a selectivefailure of the antirreflux barrier or different gastric acid secretion and/or distribution. In ambulatory conditions, a significant proportion of acid reflux is belch related. * ~==,==,,= :~s=es,~===~

Total reflux eve~Cz/24h

Trod acid (pH below 4)

Minor acid (pH above 4)

Non-acid (no pH tin)p)

Nocmat GF.RD

395(32-53)_ 46(25-64)

13(6.5-21) 215(9-35)*_

12.5(8-19) 10.5 (7-16):1:

13(8.5-19) 11 (9-16):[;

m

604

Oxidative Damages Are Critical In Pethogenesis Of Reflux Esophagitis; Implication Of Acgmddaats In Its Treatment Tas-Young Oh, DongA Pharm Research,Yongin South Korea; Jeong-Sang Lee, Seoul National Univ, Seoul South Korea; Byoung-OkAhn, Hyeon Cho, Won-Bae Kim, DongA Pharm Research,Yongin South Korea; Young-Bae Kim, Ajou Univ, Dept of Pathology, Suwon South Korea; Kwang-Hyun Ko, Sung-Won Cho, Ajou Univ, Dept of Gastroenterology,Suwon South Korea

Mechanical Charactedntics Of The EGJ After FuodolollcaNon Companld To Nonnof Subjects And GERD Patients Jennifer Curry, Guoxiang Shi, John E. Pandolfino, Raymond J. Joehl, Northwastem Univ Medical Sch, Chicago, IL; James G. Brasseur, PennState Univ, University Pad(, PA; Peter J. Kahrilas, Northwastem Univ Medical Sch, Chicago, IL Background: This study compared the mechanical characteristics of the esophagogastric junction (EGJ) of normal subjects (NL), patients with gastroecopbagealreflux diseaseand a hiatal hernia (GERD), and post-fundoplication patients (FP). Methods: Eight NL (4 male, 2459 yrs), 9 GERD patients (3 male, 22-57 yrs), and 14 FP patients (5 male, 30-68 yrs) ware studied with concurrent manometryand fluoroscopy during barostat distention accomplished by a cylindrical barostat bag mounted on the manometric catheter straddling the EGJ. The minimal intra-bag pressure required to open the EGJ during the inter-swallow periods was determined (OP). Barium swallows were imaged fluoroscopically at intra-hag pressures of 10, 15, 20, 25, 30, and 35 mmHg to determine the corresponding EGJ diameters during deglutitive relaxation and the pressure neededto obtain a 1 cm EGJ diameter (Plcm). The narrowest diameter and the length of the narrowed EGJ segment were measured. Results: EGJ opening pressure was significantly lower and deglutitive EGJ opening diameter was significantly greater in GERDcompared to NL and FP at all intra-bag pressures. EGJ opening pressure and opening diameter during deglutitive relaxation were comparable between NL and FP but EGJ length was 54% longer in FP and 23% shorter in GERD compared to NL. Conclusions:The EGJ of GERDpatientswas more distensiblethan NL. Fuodoplicationrestores the OP and distensibility of the EGJ to a level similar to normal. However,the EGJ was 54% longer than NL after FP. Since trans-EGJ flow is related to EGJ length and EGJ diameter to the 4th power, these findings suggest that flow rate may be increasedmore than 8 fold during relaxation in the GERD patients and decreasedby about 50% in the FP patients.

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Background.The facts that the severity of reflux esophagitis cannot be accurately predicted on the basis of acid exposure and acid suppressiontreatment is not enough for the complete healing suggestedother damagingfactors might be involved in pathogenesisof reflux esophagitis. Aim~ The present study was designed to evaluate the oxidative stress as the major pathogenicfactor of reflux esophagitisand the importance of antioxidant in treatment of reflux esopbagitis. Method~.Reflux esophagitis was induced by the insertion of small caliber ring (3mm in diameter) into the duodenum lcm distal to the ligament of Treitz in rats. Results:DA9601, a novelantioxidantsubstance,attenuatedthe gross esopbagitissignificantly comparedto that treated with ranitidine, histamine-2 receptor antagonist (H2-RA), in a dose-dependent manner. Severe,hemorrhagic,and longitudinal ulcerationswere developedin H2-RApretreated group, whereas mildly scattered erosions were observed in antioxidant-pretreated group. Significantly increasedamounts of malondialdehyde(MDA), increased NF-KB activation, and the mucosal depletion of reduced glutathione (GSH) were observed in the esophagusof reflux esophagitis. H2-RA treatment didn t affect the levels of GSH and MDA, whereas DA-9601 attenuatedthe decrement of the GSH levels and significantly decreasedlipid peroxides in the esophagus.Antioxidants treatment showed significant reductions in the activation of NF-KB, inflammation-associated transcription factor, especially p50 component in accordance with significant higher levels of NF-KB repressor, IgB,z expression. Conclusion:Oxygen-derived free radicals seem to be one of the important mediators in generation of reflux esophagitis, which suggests that the combination of antioxidant and anti-secretory medications will be ideal and beneficial in the prevention and treatment of reflux esophagitis than currently prescribed anti-secretory treatment alone.