Endothelin (ET-1) mediated effects on hamster small intestine submucosal microcirculation in response to hemorragic shock

normoxia (21% oxygen, 5% carbon dioxide and 74% nitrogen) instead of anoxia. DNA tragmentation (Cell Death Detection Kit) and LDH release were measured at different times after anoxia or reoxygenation in the presence or absence of PPI (lansoprazole, omeprazole, . rabeprazole) as indices ol apoptosis and necrosis, respectively, in antother experimental series, caspase 3 activity and cy'tochrome C activation were measured after anoxia Results: Exposure to anoxia significantly increased DNA fragmentation in HAEC, but did not affect LDH release. Activation of cytochrome C and caspase-3 significantly increased prior to the augmentation of DNA hagmentation. PPI inhibited DNA fragmentation and the increase in both cytochrome C and caspase-3 activity in a dose-dependent manner. LDH release from HAEC increased in the period of reoxygenation after anoxia and PPI also inhibited the increase in reoxygenation-induced LDH release. Conclusion: PPI reduced anoxia-induced apoptosis in HAEC by inhibiting the activation of cytochrome C and caspase-3 as well as reoxygenation-induced necrosis. These results suggest that PPI may have protective effects on gastrointestinal damage induced by anoxia/reoxygenation through inhibiting endothelial cell injuries.

S1071

Endothelin (ET-1) Mediated Effects on Hamster Small Intestine Submucosal Microcirculation in Response to Hemorragic Shock P H. MacDonald, J. D. Mewburn, C. E. King-Vanvlack We have recently demonstrated that ET-1 sutfusmn reduced m~crocirculatory blood flow due to both a vasoconstrictor action in higher order vessels and an inflammatory effect in lower order vessels in the small intestinal microcirculation. However, it is unclear if these reported effects of ET- I also occur in physiological conditions which stimulate the production of ET- 1. The purpose of the current study was to deternune if hemorrhagic shock (a condition associated with increased ET-1 levels) resulted in an ET-1 mediated effect on the intestinal microvascular flow. In the current experiments, videomicroscopy was used to study ileal intestinal submucosal microvascular blood flow in anesthetized hamsters. Vessel diameter and red blood cell velocity of third order arterioles and venules were measured and blood flow was calculated. In Group 1 (n=6), nreasures were obtained under control conditions, 30 rain after induction of hemorrhagic shock (MAP ~ 50 mmHg) and then 30 rain after suffusion with the Endothelin-A receptor antagonist BQ-123 (105M) dunng hemorrhagic shock. In Group 2 (n = 3) similar measurements were made except that BQ-123 was administered before the induction of hemorrhagic shock. Group 3 (n = 2) served as a time control and measurements were made under control conditions and then 30 and 60 minutes alter induction of hemorrhagic shock Arteriolar and velmlar blood flow decreased without any change in vessel diameter at 30 rain of hemorrhagic shock and remained at this level at 60 rain of shock (Group 3). This fall in arteriolar blood flow at 30 rain of hemorrhagic shock was partially restored with BQ-123 (Group 1). "When BQ-123 was admimstered before hemorrhage (Group 2), blood flow was not ahered with BQ- 123 suffusion and the subsequent induction of hemorrhagic shock had no effect on arteriolar flow. Venutar responses were similar to those observed in the arterioles. These findings demonstrate that the reduction in blood flow in third order submucosal arterioles and venules during severe hemorrhagic shock is partially mediated by ET-1. The ET-1 induced reduction in blood flow occurred in the absence of significant vasoconstriction, suggesting that the pro-inflammatory actions of ET-1 were responsible for the decrease in blood flow; a finding not unlike our pre.aous findings with exogenous ET-I. administration. Supported by Canadian Institutes for Health Research.

S1069

MAdCAM-1 Expression in Human Intestinal Microvascular Endothelial Cells (HIMEC) is Regulated by the PI3K/Akt and NFKB Pathways Hitnshi Ogawa, Parvaneh Rafiee, Pamela Fisher, Nathan Johnson, Jan Heidemann, Mary Otterson, David G Binion Background and Aims: Microvascular endothelial expression of the mucosal addressin cell adhesion mdecule-1 (MAdCAM-1) plays a critical role in the recruitment of circulating leukocytes into the intestinal mucosa, in addition to ICAM-1 and E-selectin, MAdCAM-1 expression is increased in inflamed IBD mucosa, however the molecular mechanisms underlying the regulation of MAdCAM-1 expression in human intestinal endothelial cells are presently not defined. Using primary cultures of microvascular endothelial cells isolated from human intestinal mucosa, we defined the signaling mechamsms underlying MAdCAM-1 expression. Methods: HIMEC were used in experiments between passages 8-14. HIMEC monolayers were activated with TNFet or LPS after culture for I, 4, or 7 days, or at defined cellular densities. RT-PCR analysis was used to detect MAdCAM-1, ICAM-1 or E-selectin gene product. Western blotting was performed to examine PI3K/Akt activation by TNFa or LPS. PI3K inhibitor (LY 200539) and NFKB inhibitor (SNSO) were used to define signal transduct ion pathways underlying MAdCAM-1, ICAM-1 or E*selectin expression. Results: MAdCAM1 mRNA expression in response to TNFc~or LPS was increased following prolonged culture after the monolayers reached confluence. Increased MAdCAM-1 expression was also seen ni HIMEC cultures plated at higher cellular densities, which corresponded with increased Akt phosphorylatinn by TNF~ or LPS In marked contrast, ICAM-1 or E-selectin mRNA or protein expression was not affected by culture duration or cellular density. MAdCAM-1 expression was inhibited by both LY 200539 and SN50, while ICAM-1 or E-selectin expression was inhibited by SN50 but not by LY200539. Conclusions: MAdCAM-1 expression in human gut specific microvascular endothelial cells is induced through both the PI3K/Akt and the NFKB pathways, while ICAM-1 and E-selectin were predominantly regulated by NFKB. Longer culture duration and higher cellular densitites in vitro increased MAdCAM1 expression in response to TNFa or LPS, which corresponded with increased PI3K/Akt activity. These results indicate that cdl-cefl interaction plays a critical role in the regulation of MAdCAM-1 expression through ~he PI3WAkt pathway. These results are in marked distinction from the signaling pathways which lead to expression of ICAM-1 or E-selectin, and targeting P13R/Akt may be considered a therapeutic target for modulating MAdCAM1 expression in wvo

$1072 Physiologic Expression of Cox2 and PGE2 in Human Intestinal Microvessels and Isolated Endothelial Cells (HIMEC) Parvaneh Rafiee, Ossama A. Haloum, Keith T. Wilson, Mary Otterson, David D. Gutterman, David G, Binion Background and Aims: Gut microvascular endothelial cells play a critical role in intestinal physiology and homeostasis, through their regulatory effect on vasorelaxation and tissue perfusion. We characterized the ability of H1MEC to express cyclo-oxygenase 2 (COX2), as well as production of the vasoactive prostaglandin PGE2. We examined the physiologic role of cyclooxygenase in normal microvascular dilation in response to acetylcholine (Ach). We also examined the effect of cyclosporine A (CsA), a ealcnieurin inhibitor known to contribute to vascular dysfunction in the setting of chronic transplant rejection, on HIMEC expression of COX2 as well as intact microvesseI relaxation Methods: HIMEC monolayers passage 814 stimlnated with cytokines and LPS, were used to examine COX2 expression by western blotting and fluoresence microscopy. PGE2 production in conditioned media was examined using ELISA.Effect of Ach on isolated intestinal submucosal arterioles (100-200 mm diameter) was determined. Diameter of isolated eannulated vessels was measured by videomicroscopy, v,nth and vctthout treatment with the COX inhibitor indomethacin. CsA was used to pretreat HIMEC monolayers as well as whole vessels Results: HIMEC expressed low levels of COX2 by western blotting, which was significantly increased 3 fold following activation with TNF/I_P8 or 2 fold with VEGF, but not TNF, IL-1 or I1_-8alone. COX2 expression in response to VEGF was not sustained beyond 6h, while TNF/I_PSinduced expression remained etevated at 24h. Immunolocafization revealed cytosolic and perinuclear expression of COX2 in H1MEC. Unstimulated HIMEC monolayers produced 677 -+ 183 pg/ml (mean -+SE) PGE2/lxl06 cells, which increased to 29,725 -+6155 pg/ml after 24h (n = 12; p = <0,O01) Intact small and large bowel microvessels exhibited a 40% reduction in Ach induced vasodilation following treatment with indomethacin (Max dilation 50 + 10% vs control 90 +-3%, n = 7), which was similar to CsA's inhibitory effect. CsA treatment of HIMEC monolayers inhibited COX2 expression induced by both TNF/LPS and VEGF. Conclusions: H1MEC express COX2 in response to inflammatory and angiogeinc stimuii, which corresponds with increased PGE2 production Gut microvascular endothelial ceils produce large amounts of the vasoactive prostaglandin PGE2. and intestinal microvessels rely heavily on COX dependent mechanisms for vasoddation CsA exerted potent inhibitory effects on COX2 expression which inhibited normal vasorelaxation responses

S1070

Specific Serum Antibodies are Involved in H. pylori-lnduced Platelet Aggregation Paul A Corcoran, Stephen W. Kerrigan, Dermot Cox, John C. Atherton, Desmond J. Fitzgerald, Frank E. Murray, Michael F. Byrne Background: Clinical studies have suggested an association between cardiovascular disease and infection with H. pylori. We have described strain specific H. pylon-induced platelet aggregation that involves platelet glycoprotein Ib and von Willebrand factor (vW0. We examined the role of H. pylon antibodies in this aggregation. Methods and Results: Addition of pooled immunoglobin (lg) and fibrinogen along with H. pylori strain 60190 (coated in vWf) to washed plateiets Ied to piateiet aggregation (60 + 4%, n = 3) Antibodies involved were specific to H. pylori as bactena incubated in pooled lg and subsequently washed could support platelet aggregation (49 -+ 7%, n = 3). To confirm this, pooled Ig was incubated with H. pylon or Streptococcus sanguis. Bacteria were removed by centrifngatinn and the depleted lg used in aggregation assays. H. pylori-depleted Ig showed greatly reduced platdet aggregation to H pylon (13_+9%, n = 3, p<0.01) while the S. sangnis-depleted Ig was not affected (54 _+3%, n = 3, p = NS). All 20 plasma donors were tested for H. pylori seropositivity using a H pylon ELISA All 4/4 of the positive volunteers consistently aggregated in response to H pylon while only 2/15 of the H. pylori negative donors aggregated consistently (p = 0039, Fisher's Exact Test). The relative risk of H pylonn positivity for aggregation was 7.5 (CI 2.0-27.3). Conclusions: H. pylon-induced pLatelet aggregation requires binding of H. pylori-specific lgG This pro-aggregatory phenotype is not unique to H. pyiori and has been described with S. sanguLsand P. gingivalis. Exposure to oral bacteria such as S. sanguis can lead to infective endocarditis and, in the case of P gingivalis, to enhancement of atherosclerosis in animal models. Repeated exposure to pro-aggregatory bacteria such as certain strains of H. pylon may not cause coronary artery disease but could accelerate the progression of the disease Local platelet effects may also contribute to the pathogenesis of H. pylori-associated peptic ulcer disease

AGA Abstracts

$1073 5-HT3 receptor antagonists, alosetron and cilansetron, impair mesenteric blood flow in rats Peter Hoher, Evelin Painsipp, Eckhard Weber, Hans-Juergen Pfannkuche Background: 5-HT3 receptor antagonists, alosetron and cilansetron, have been developed for the treatment of irritable bowel syndrome (IBS) with diarrhea, whereas tegaserod, a partial 5-HT4 receptor agomst, has been launched to treat IBS with constipation. While tegaserod has been shown to be safe, treatment of IBS patients with alosetron has been associated with cases of ischemic colitis. This study was designed to evaluate the effects of tegaserod, alosetron and cilansetron on mesenteric hemodynamics in the rat Methods: Phenobarbital-anesthetized rats were instrumented (i) to record mean arterial blood pressure (MAP) and heart rate (HR) from a carotid artery, (ii) to measure blood flow in the superior mesenteric artery (MBF) via the ultrasound transit time shift technique and (iii) to measure phasic and tonic motor activity in the colon via intraluminal pressure (1LP) recording. Mesenteric vascular conductance (MVC) was calculated as MBF divided by MAP. After a 2

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