AG

AA

bst

ract

sPatients experienced a mean increase of 25.52% (p<0.001) in CFA compared to baselinevalues. Zentase also improved vitamin K status and malabsorption signs and symptoms,including a statistically significant reduction in stool frequency (p<0.001) and fewer softstools (p<0.001). Signs and symptoms of malabsorption improved with Zentase treatmentregardless of EPI severity, including in patients with CFA values > 80% on placebo. Halfof the patients treated with Zentase had a CFA >90% and 91% of the patients treated withZentase had a CFA >80%. A positive correlation was observed between Zentase dose andresponse, particularly in patients with abnormal stool at baseline. Conclusion: In this PhaseIII study, Zentase, a novel PEP, was safe, well tolerated and effective in the treatment ofEPI in cystic fibrosis, with clinically and statistically significant improvements in CFA, CNA,and malabsorption signs and symptoms in the absence of any concomitant treatment affectinggastrointestinal motility and pH. The therapeutic benefit of Zentase was seen in patientswith mild EPI (CFA values >80% on placebo), as well as in patients with moderate andsevere disease.

T1887

Red Wine Polyphenols Reduce Postprandial Lipid Peroxidation END ProductsAbsorptionMoshe Ligumsky, Shlomit Gorelik, Joseph Kanner, Ron Kohen

Dietary fat peroxidation and formation of toxic end products has been implicated in thepathogenesis of atherosclerosis (1), while dietary polyphenols may attenuate this process(2). We have shown that red meat peroxidation is inhibited by polyphenols in simulatedgastric milieu, but postprandial dietary lipid oxidation and the effect of polyphenols In Vivohave not been explored. Aim: To investigate, in rat, postprandial lipid peroxidation in thestomach following fat-rich meal and the effect of red wine polyphenols. Methods: #1: Afterovernight fasting and collecting blood samples, male rats (220-250g) were fed with one of2 meals (grilled at high temp): Meal A=red turkey meat cutlets (control) or B=meal A+alcohol-free red wine containing 3µmoles polyphenols (test meal). Rats were sacrificed 90min later, plasma samples were collected and gastric content was weighted. #2: A separategroup of rats was fed with homogenized, heated red turkey meat by gavage. Under anesthesiathe pylorus was cannulated for collection of gastric content every 15-20 min. Oxidationproducts, malondialdehyde (MDA) were determined by measuring thiobarbituric acid react-ive compounds and hydroperoxides (LOOH) by the FOX2 method. Statistical analysis wasperformed by analysis of variance followed by Student's-Newman-Keuls test. Results: #1.Meal A (n=14) and Meal B (n=11) exhibited similar oxidative capacity at the baseline(216.2±34.5 µM and 202.9±23.2µM for LOOH and 79.5±13.0 µM 85.9±14.9µM for MDA,respectively; mean±SD). 90' after eating, LOOH capacity in the residual gastric contentdecreased to 70±51.0µM in meal A, but greater reduction (*19.5±22.6µM), was observedfor meal B (*p<0.05). Similar trend was seen for MDA. #2. In rats (n=3) fed by gavage,MDA level in the gastric content gradually increased to 2.3 folds at 120'. Similar increasewas observed for LOOH. 3. Postprandial (90') MDA plasma levels increased by 50% frombaseline following Meal A (n=8) while it was reduced by 34% following polyphenol-enrichedmeal B (n=6). Conclusions: 1.During meal, partially oxidized food presumably undergoeslipid peroxidation in the stomach and formation of toxic lipid oxidation end products whichare absorbed into the plasma. 2. Wine polyphenols may attenuate toxic lipid derivativesformation during meal and decrease their absorption into the plasma. 3. By counteractingtoxic lipid compounds formation and diminishing their postprandial absorption (3), dietarypolyphenols may have an important role in the protection against development of atheroscler-osis. References: 1.Fruhwirth et al. BBA 2007; 2. Lapointe et al. J Nutr Biochem 2006;3.Gorelik et al. FASEB J, in press

S1887a

Inhibition of Intestinal Heme Transport By the Metalloporphyrin Cr-TmpMark T. Worthington, Jay Umbreit, Wyeth Callaway, Jerry Bommer

Background: Heme is the most important and bioavailable source of dietary iron; requiredfor oxygen transport, mitochondrial oxidative phosphorylation, and xenobiotic metabolism.For decades, it was assumed that heme passively diffused across biological membranes: earlystudies performed using CO-modified heme coordinated to the iron adduct and eliminatedthe intrinsic metal character of this metalloporphyrin. Heme and inorganic iron can be quitetoxic, therefore strict regulatory processes exist in mammalian cells to minimize damage.Previous studies from our laboratory (AJP-GI 280:G1172-77 (2001) and Cell 118:757-766(2004)) and others clearly demonstrated that not only does heme movement across biologicalmembranes require specific transport proteins, but these proteins permits additional levelsof regulation. Since there is no mammalian pathway to secrete excess iron, when thisregulation is abnormal, there is the potential for severe tissue damage and death, such asin the iron overload disease hereditary hemochromatosis (HH). In HH, uncontrolled ironuptake leads to end-organ damage such as cirrhosis, cardiac failure, diabetes, and arthritis,requiring lifetime phlebotomy. Earlier studies using a heme oxygenase (HO) inhibitor, thestep immediately after membrane uptake, led to iron deficiency anemia in normal hosts andnormal rats, but is associated with photosensitivity dermatitis. A specific oral heme transportinhibitor would be a promising drug. Aim: To identify soluble, nontoxic inhibitors ofintestinal heme uptake that could be used in HH to reduce the need for phlebotomy. Results:We screened over 40 substituted metalloporphyrins to identify compounds that effectivelyinhibit apical heme uptake in IEC-6 rat intestinal epithelial cells. One of these compounds,Cr (III) meso-tetra(N-Methyl-4-pyridyl) porphine * 4 Tos (Cr-TMP), inhibits heme uptakewith an IC50 of 3 uM. The choice of TMP metal ligand was critical, with IC50 from smallestto highest Cr (III) < Fe(III) < Ni, Cu < Zn < Ga < Eu < Sn (IV). No toxicity of Cr-TMP wasseen with IEC-6 intestinal epithelial or K562 erythroleukemia cells. Cr-TMP is poorlyabsorbed in IEC-6 and K562 cells, and the bulky porphyrin substituents make effects onHO unlikely. A homologous compound with only three N-Methyl-4-pyridyl porphine sidechains (Cr-TriMP) is as effective as Cr-TMP in inhibiting transport in IEC-6 cells. In rats,Cr-TMP with radioactive heme and the bile salt taurodeoxycholate in closed duodenal loopsprevents near complete absorption of iron from the heme at both 50 uM and 200 uMconcentration. Assessment: Cr-TMP is promising as an inhibitor of intestinal heme transport.

T : 11501$$CH204-02-08 16:47:13 Page 584Layout: 11501B : e

A-584AGA Abstracts

T1888

Tocotrienols Destabilize the Mitochondrial Electron Transport Chain (Metc)Setting Off Mitochondrial Oxidative Stress, BAX Activation, and Autophagicand Apoptotic Cell Death in Activated Pancreatic Stellate Cells (PSCs)Mariana Rickmann, Ana González, Xavier Molero, Eva C. Vaquero

We have previously shown that palm oil tocotrienols cause apoptosis and autophagy inactivated (not quiescent) PSCs by targeting the mitochondrial permeability transition pore.The mETC activity is coupled to a transmembrane electrochemical gradient (DeltaPsim).mETC destabilization may result in DeltaPsim collapse, ROS overproduction, and cell death.Herein we study mitochondrial oxidative stability in PSCs in response to tocotrienols andits impact in death signaling. METHODS: Plastic-cultured (activated) and Matrigel-cultured(quiescent) rat PSCs were treated with palm oil tocotrienol-rich fraction (TRF) for up to24h. DeltaPsim, iROS (intracellular) and mROS (mitochondrial) were determined withDiOC6(3), DCFH-DA, and MitoSOX using FACS analysis; apoptosis was assessed by DNAfragmentation, caspase activities, and cytochrome c release; autophagy by LC3 processing;Bax activation by crosslinking and immunoblot; mitochondrial morphology by TMRM usingconfocal analysis. RESULTS: Quiescent PSCs showed lower DeltaPsim, iROS and mROSthan activated PSCs. TRF inhibited ROS formation, did not alter DeltaPsim, and caused nodeath in quiescent PSCs. On the contrary, beginning 30 min after addition, TRF triggeredan intense and persistent mitochondrial depolarization in activated PSCs, and caused astriking mitochondrial network transition from filamentous to round fragmented structures.Subsequent earliest detection time of TRF-induced events were as follows: mROS increase(6 h), autophagy (8 h), iROS increase (14 h) and apoptosis (18 h). N-acetyl-cysteine, tiron,GSH-EE (hydrophilic antioxidants), and α-tocopherol (lipophilic antioxidant) effectivelyprevented TRF-induced iROS, but only α-tocopherol precluded mROS overproduction. Inaddition, α-tocopherol largely reduced (by 60%) TRF-induced apoptosis, in contrast tohydrophilic antioxidants (20% apoptosis reduction). However, α-tocopherol did not preventDeltaPsim collapse nor impeded TRF-induced autophagy, suggesting that sustained com-promised DeltaPsim promotes autophagy in PSCs. TRF activated Bax (conformational changeand dimerization) and this effect was greatly reverted by α-tocopherol. Pre-treatment witha Bax channel blocker significantly diminished (by 60%) apoptosis in TRF treated cells, butit did not revert mitochondrial depolarization nor prevented death by autophagy. CONCLU-SIONS: Tocotrienols disrupt mETC in activated PSCs discerning apoptotic and autophagicdeath outcomes by triggering persistent mitochondrial depolarization, mROS formation, andBax activation. Induction of selective mitochondrial PSC destabilization may be consideredas a novel therapeutic approach for pancreatic fibrosis.

T1889

TGF-β Regulates Nerve Growth Factor (NGF) Expression in PancreaticStellate Cells By Activation of the Alk-5 PathwayStephan L. Haas, Robert Jaster, Eliza Wiercinska, Haristi Gaitantzi, Ralf Jesnowski,Johannes M. Löhr, Manfred V. Singer, Steven Dooley, Katja Breitkopf

Background: Chronic pancreatitis (cP) is characterized by intermittent bouts of inflammationin conjunction with severe abdominal pain. Histologically proliferation of intrapancreaticnerves was determined in cP. Nerve growth factor (NGF) is the prototypical member of theneurotrophin family which promotes neurite outgrowth and pain. There is accumulatingevidence that NGF has complex cytokine-like and immunomodulatory functions. Effects ofNGF are mediated by the low-affinity NGF receptor p75NTR and the high-affinity tyrosinekinase receptor TrkA. Aim: To characterize expression and regulation of NGF, TrkA, andp75NTR in primary pancreatic stellate cells (pPSC) and in an immortalized human PSC cellline (ihPSC) which we recently developed by SV40 large T antigen and human telomerasetransfection. Methods: NGF, p75NTR and TrkA expression was studied by RT-PCR, Westernblot and immunofluorescence. Time (1h, 6h, 24h) and dose-dependent regulation of expres-sion was examined after TGF-β stimulation (0.5 ng/ml; 5.0 ng/ml). Activation of the TGF-β induced Smad signaling pathways was investigated by Western blot assessing Smadphosphorylation and in reporter gene assays (CAGA-assay). Results: NGF, p75NTR and TrkAwere all expressed and induced by TGF-β in ihPSC while this was only true for NGF andTrkA in prPSC. While TGF-β stimulation led to activation of Smad2 in ihPSC as well asprPSC, only in prPSC parallel activation of Smad1 and expression of the TGF-β receptorALK1 was detected. The TGF-β mediated induction of NGF secretion was blocked byinhibiting the ALK5/Smad2/3/4 pathway using SB431542 - a chemical compound drug thatblocks ALK5, the classical TGF-β type I receptor. Conclusions: Early primary PSC do expressNGF and TrkA. Expression is upregulated by the most potent profibrogenic mediator TGF-β via ALK5. In contrast to a recently established PSC cell line, primary PSC respond toTGF-β by activation of both the ALK1/Smad1/5/8 and ALK5/Smad2/3/4 signaling cascade.

T1890

Bone Marrow-Derived Pancreatic Stellate Cells in MiceTakashi Watanabe, Atsushi Masamune, Kazuhiro Kikuta, Tooru Shimosegawa

Background & Aim: Activated pancreatic stellate cells (PSCs) play a crucial role in pancreaticfibrosis. In response to pancreatic injury or inflammation, they are transformed (“activated”)from their quiescent phenotype into myofibroblast-like cells, which actively proliferate,express alpha-smooth muscle actin (alpha-SMA), and produce extracellular matrix compon-ents. The origin of PSCs has been thought to be peri-acinar or peri-ductal location. Recentstudies have suggested that bone marrow-derived cells might participate in regenerationprocesses of various organs. This study aimed to clarify the contribution of bone marrow-derived cells in pancreatic fibrosis. Methods: Female C57BL/6J mice received 1x10^7 bonemarrow cells of age-matched male C57BL/6J mice one day after total body irradiation withbone marrow lethal dosage of 7 Gy. Hematologic reconstitution was confirmed by detection ofY chromosome in the spleen of recipient mice. Eight weeks after bone marrow transplantation,chronic pancreatitis was induced by administration of six intra-abdominal injections ofcaerulein (50 ug/kg) at 1-hour intervals, three days weekly, for the total of eight weeks. Thecontrol group received 0.9% saline. Thereafter, all mice were sacrificed and pancreas tissues