EXPEDITED ARTICLE
Inhibition of poly(ADP-ribose) polymerase attenuates inflammation in a model of chronic colitis

Humberto B Jijon1, Thomas Churchill2, David Malfair3, Andreas Wessler4, Laurence D Jewell4, Howard G Parsons1, and Karen L Madsen3

1 University of Calgary and 2 Surgical Medical Research Institute, 3 Division of Gastroenterology, and 4 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta T6G 2C2, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Crohn's disease is a chronic disease characterized by oxidant-induced tissue injury and increased intestinal permeability. A consequence of oxidative damage is the accumulation of DNA strand breaks and activation of poly(ADP-ribose) polymerase (PARP), which subsequently catalyzes ADP-ribosylation of target proteins. In this study, we assessed the role of PARP in the colitis seen in interleukin (IL)-10 gene-deficient mice. IL-10 gene-deficient mice demonstrated significant alterations in colonic cellular energy status in conjunction with increased permeability, proinflammatory cytokine release, and nitrosative stress. After 14 days of treatment with the PARP inhibitor 3-aminobenzamide, IL-10 gene-deficient mice demonstrated normalized colonic permeability; reduced tumor necrosis factor-alpha and interferon-gamma secretion, inducible nitric oxide synthase expression, and nitrotyrosine levels; and significantly attenuated inflammation. Time course studies demonstrated that 3-aminobenzamide rapidly altered cellular metabolic activity and decreased cellular lactate levels. This was associated with normalization of colonic permeability and followed by a downregulation of proinflammatory cytokine release. Our data demonstrate that inhibition of PARP activity results in a marked improvement of colonic inflammatory disease and a normalization of cellular metabolic function and intestinal permeability.

interleukin-10; 3-aminobenzamide; inflammatory bowel disease; intestinal permeability


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

UNDER NORMAL PHYSIOLOGICAL conditions, the intestinal epithelium acts as a selective barrier, allowing uptake of nutrients and water but limiting the passage of luminal antigens into the lamina propria. The barrier function of the intestine is regulated at the level of the tight junctions close to the apical surface of epithelial cells (30). Tight junctions are dynamic, energy-requiring protein structures that can be modulated by bacterial products (12, 14, 15), dietary constituents (2, 23), immunological factors (26, 36), alterations in cellular energy status (29, 30, 44), and pH (31, 39). Disruption of tight junctions leads to increases in epithelial permeability and abrogated barrier function.

The lumen of the intestine contains bacteria, bacterial products, and dietary antigens capable of initiating and sustaining inflammation. Disruption of the epithelial barrier, particularly in the crypt region, could lead to aberrant antigen uptake, an uncontrolled activation of the mucosal immune system, and subsequent intestinal inflammation (11). Patients with Crohn's disease have increased intestinal permeability, either as a primary defect or as an acquired defect secondary to intestinal inflammation (8, 16, 35). This defect in intestinal barrier function has been postulated to play a role in the exacerbation of intestinal inflammation in Crohn's disease by allowing increased antigen uptake and a continuous stimulation of the mucosal immune system.

The disruption in barrier function of the intestine during active inflammation has been linked to the generation of reactive oxygen and nitrogen species and resultant tissue injury (13). High levels of superoxide (SO) and nitric oxide (NO) have been described in areas of active inflammation (13). These can either directly effect tissue injury or react together to produce the potent oxidant peroxynitrite (ONOO-) (4). Peroxynitrite can cause oxidative damage and DNA strand breakage within epithelial cells (41). One consequence of oxidative damage is the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) (22). This enzyme acts to transfer the ADP-ribose moiety of NAD+ to various chromatin proteins (43). Chronic activation of PARP can potentially lead to a depletion of cellular NAD+ and, because NAD+ is necessary for ATP synthesis, a decline in cellular ATP levels and potential cell dysfunction (43). Indeed, in vitro exposure of intestinal epithelial monolayers to a peroxynitrite donor results in PARP activation, ATP depletion, and decreased transepithelial resistance (19).

Currently, strategies for treatment of inflammatory bowel disease remain focused on suppression of the cellular inflammatory response. However, these approaches to anti-inflammatory therapy, including the use of monoclonal antibodies against tumor necrosis factor (TNF)-alpha or the use of the anti-inflammatory cytokine interleukin (IL)-10, remain suboptimal. Because the disrupted mucosal barrier function seen in patients with inflammatory bowel disease almost certainly acts to exacerbate intestinal inflammation by allowing increased antigenic uptake, we predict that therapy aimed at correcting such a permeability defect would have a beneficial effect in treating chronic inflammation.

In the present study, we have assessed the role of PARP in the increased permeability that is associated with chronic, nonresolving colitis that develops spontaneously in the IL-10 gene-deficient mouse (5, 28). Here we demonstrate that pharmacological inhibition of PARP activity results in a rapid normalization of cellular metabolic function and intestinal permeability followed by a dramatic improvement in colonic inflammatory disease.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Chemicals of reagent grade were obtained from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Napean, ON, Canada). The radioisotopes [14C]polyethylene glycol and D-[3H]mannitol were obtained from New England Nuclear (Boston, MA). ELISA kits used for the measurement of TNF-alpha and interferon (INF)-gamma were obtained from Medicorp (Montreal, PQ, Canada). PARP activity was analyzed using the PARP activity assay kit obtained from Genzyme Diagnostics (Cambridge, MA).

Animals

Homozygous IL-10 gene-deficient mice, generated on a 129 Sv/Ev background, and 129 Sv/Ev controls were housed under specific pathogen-free conditions. All provisions for the facility were autoclaved. Nonautoclavable supplies were sprayed with disinfectant and introduced through a HEPA-filtered air lock. Mice were housed in microisolator cages with tight-fitting lids containing a spun polyester fiber filter. In sentinel BALB/c mice, bacterial cultures, parasitological examinations, serological tracking profiles, and histological stains were negative for known murine viral and bacterial pathogens, indicating that the barrier was intact.

Experimental Groups

Chronic. IL-10 gene-deficient mice were treated with the PARP inhibitor 3-aminobenzamide (20 mg/kg) via an intraperitoneal injection for 14 days, starting at 8 wk of age (a time when colitis is firmly established). A vehicle-treated group received 3-aminobenzoic acid, an inactive structural analog of 3-aminobenzamide with no inhibitory effect on PARP activity (3). Food intake was monitored, and weight gain was recorded over the 14-day treatment period. Colons from mice were assessed histologically, and colonic permeability, TNF-alpha and IFN-gamma secretion, and nitrotyrosine, ATP, ADP, AMP, and lactate levels were measured. In addition, wet weights of heart, lungs, liver, kidneys, and small and large intestine were recorded, as were small and large intestinal lengths.

Acute. Three-week-old IL-10 gene-deficient mice do not demonstrate any histological signs of inflammation or any neutrophilic influx into the lamina propria but do show increased colonic permeability (28). To determine the acute effects of 3-aminobenzamide treatment in the absence of significant ulceration, IL-10 gene-deficient mice were injected with either saline (0.2 ml/kg) or 3-aminobenzamide (20 mg/kg i.p. in 0.2 ml/kg saline). At 2 and 4 h following injection, separate groups of mice were anesthetized for measurement of colonic permeability, TNF-alpha and IFN-gamma secretion, or cellular ATP and lactate levels.

Intestinal Histological Assessment

Animals were killed using pentobarbital sodium (160 mg/kg). Segments of colon were harvested, fixed in 10% phosphate-buffered formalin, paraffin embedded, sectioned at 4 µm, and stained with hematoxylin and eosin for light microscopic examination. The slides were reviewed independently by two pathologists in a blinded fashion (A. Wessler and L. D. Jewell) and assigned a histological score for intestinal inflammation using a scheme adapted from Saverymuttu et al. (40) and detailed in Table 1. Histological grades represent the numerical sum of four scoring criteria: mucosal ulceration, epithelial hyperplasia, lamina propria mononuclear infiltration, and lamina propria neutrophilic infiltration.

                              
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Table 1.   Grading scheme for histological intestinal injury

Intestinal Permeability Measurements

In vivo perfusion. On the day of study, mice were premedicated with atropine (0.2 mg/kg) followed by an intraperitoneal injection of Hypnorm (25 mg/kg) and midazolam (12.5 mg/kg). In vivo absorption was measured by a single-pass perfusion technique, as previously described (32). In brief, the entire colon was isolated and cannulated at the proximal and distal ends. The gut was flushed with isosmotic Tyrode buffer (in g/l: 8 NaCl, 0.2 KCl, and 0.33 NaH2PO4, pH 7.4) to clear luminal contents. The segment was then perfused with test solution containing 5 g/l polyethylene glycol 4000 and 1 mM D-mannitol prepared in Tyrode buffer and radiolabeled with [14C]polyethylene glycol (10 µCi/l) and D-[3H]mannitol (100 µCi/l). Preliminary experiments determined that polyethylene glycol 4000 was not absorbed in either control or IL-10 gene-deficient mice (data not shown). The solution was perfused at a constant rate of 0.2 ml/min, and body temperature was maintained at 37°C. Intraluminal hydrostatic pressure was constantly monitored. After a 30-min equilibration period, six consecutive 10-min perfusion samples were collected from the distal site. The samples were weighed, and aliquots were taken for liquid scintillation counting. After completion of the procedure, animals were killed and the perfused segment of intestine was removed; its length was measured, and then it was dried for determination of dry weight.

Intestinal permeability. Net water flux was calculated on the basis of differences between initial and final volumes of perfusate and by the differences between initial and final concentrations of [14C]polyethylene glycol according to the following equation
Pump Volume<IT>−</IT>{(Experimental  [<SUP><IT>14</IT></SUP>C]polyethylene glycol

÷Initial [<SUP><IT>14</IT></SUP>C]polyethylene glycol) Sample Volume}<IT>1,000</IT>

×10<SUP>−1</SUP> · g dry weight<SUP><IT>−1</IT></SUP>
Mannitol clearance was calculated by the following equation
C<SUB>probe</SUB><IT>=</IT>(C<SUB>i</SUB>V<SUB>i</SUB><IT>−</IT>C<SUB>f</SUB>V<SUB>f</SUB>)<IT>/</IT>(C<SUB>avg</SUB><IT>TW</IT>)
where Ci is the measured initial perfusate concentration, Cf is the measured final probe concentration, Vi is the measured initial perfusate volume, Vf is the calculated final perfusate volume, Cavg is (Ci - Cf)/ln(Ci/Cf), T is hours of perfusion time, and W is weight of dried gut in grams (37).

Mucosal Cytokine Secretion

Colonic organ cultures were prepared from control mice, IL-10 gene-deficient mice, and IL-10 gene-deficient mice treated with 3-aminobenzamide (20 mg · kg-1 · d-1 ip). Because of the patchy nature of colitis in IL-10 gene-deficient mice, whole colons were removed, flushed with PBS, and resuspended in tissue culture plates (Falcon 3046; Becton Dickinson Labware, Lincoln Park, NJ) in RPMI 1640 supplemented with 10% fetal calf serum, 50 mM 2-mercaptoethanol, penicillin (100 U/ml), streptomycin (100 U/ml), and in the presence or absence of lipopolysaccharide (LPS). Cultures were incubated at 37°C in 5% CO2. After 24 h, supernatants were harvested and stored at -70°C for analysis of cytokine levels. TNF-alpha and IFN-gamma levels in cell supernatants were measured using ELISA kits purchased from Biosource Cytoscreen (Montreal, PQ, Canada).

NO Synthase Activity

Colonic mucosa was homogenized on ice in a buffer composed of (in mM) 50 Tris, 0.1 EGTA, 0.1 EDTA, 12 2-mercaptoethanol, and 1 phenylmethylsulfonyl fluoride (pH 7.4). The homogenate was incubated with a cation-exchange resin (AG 50W-X8, Na+ form) for 5 min at 4°C to deplete endogenous L-arginine. Conversion of L-[3H]arginine to L-[3H]citrulline was measured in homogenates. Experiments in the presence of NADPH, without calcium and with 5 mM EGTA, were performed to determine the calcium-independent NO synthase (NOS) activity. Protein concentration was determined by the Bradford method (6).

Western Blotting

Colonic mucosa was suspended in 0.5 ml Mono Q buffer (50 mM beta -glycerophosphate, 1 mM EGTA, 2 mM MgCl2, and 0.5% Triton X-100, pH 7.2) and sonicated on ice for 30 s. Protein concentrations were determined using the Bradford method (6), and samples were diluted to the same concentration. Samples were separated by SDS-PAGE (10 µg/lane) and either silver stained (to ensure even loading of lanes) or transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked for 30 min with 3% skim milk and 20 mM Tris, 0.5 M NaCl, and 0.05% Tween 20, pH 7.4 and incubated overnight at 4°C with anti-nitrotyrosine monoclonal antibody (1:500 dilution; Upstate Biotech, Saranac Lake, NY) for nitrotyrosine blots or anti-inducible NOS (iNOS) antibody (1:1,000 dilution, rabbit anti-mouse iNOS, Upstate Biotech). Membranes were then washed 3 times with water and incubated for 2 h with goat anti-mouse antibody (Bio-Rad; 1:3,000 dilution) followed by 2 washes with water and Tris-buffered saline. Autoradiography was performed on Kodak X-OMAT AR film using a chemiluminescence kit (Lumi-light, Amersham).

Metabolite Assays

Whole colons were snap frozen using Wollenberg clamps precooled in liquid nitrogen and subsequently stored at -70°C until processing. Frozen tissue samples were weighed and then extracted at 1:5 wt/vol in 6% perchloric acid containing 1 mM EDTA. Precipitated protein in the remaining homogenate was removed by centrifugation for 15 min at 20,000 g. Acid extracts were neutralized by the addition of 3 N KOH, 0.4 M Tris, and 0.3 M KCl and recentrifuged. Aliquots of the neutralized extracts were immediately used for assays of ATP, ADP, AMP, and lactate. Metabolites were assayed enzymatically on the basis of the absorption of NADH at 340 nm, using a Dynex MRX microplate reader. Assays were performed as previously described (1).

Measurement of PARP Activity

Colons were removed, and the mucosa was scraped. The resultant tissue pellet was analyzed for PARP activity using a commercial activity assay (Genzyme Diagnostics, Cambridge, MA). Cell suspensions from mucosa were prepared by gentle physical disruption in ice-cold buffer (in mM: 50 Tris · HCl, pH 8, 25 MgCl2, and 0.1 phenylmethylsulfonyl fluoride), followed by centrifugation at 3,000 g for 5 min at 4°C. Twenty micrograms of total protein were transferred from the supernatant to microcentrifuge tubes at room temperature. The reaction was started with the addition of 1 mM NAD and 2 µCi [32P]NAD. The reaction was allowed to proceed for 1 min and then stopped by the addition of 900 µl of 20% ice-cold TCA. The contents of the reaction tube were filtered through a prewetted glass fiber filter under vacuum and washed four times with 3 ml of 10% ice-cold TCA and twice with 3 ml of 95% cold ethanol. The filters were then dried and counted. PARP activity is expressed as nanomoles per minute per microliter.

Statistical Analysis

Data are reported as means ± SE. Differences between mean values were evaluated using analysis of variance or paired t-tests where appropriate (SigmaStat; Jandel, San Rafael, CA). Specific differences were evaluated using the Student-Newman-Keuls test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chronic Treatment of IL-10 Gene-Deficient Mice

Animal profile and histological assessment. Over the 14-day treatment period, control mice and IL-10 gene-deficient mice receiving 3-aminobenzamide gained weight (Table 2). In contrast, despite having a similar food intake, IL-10 gene-deficient mice lost weight, resulting in a significantly reduced total body weight compared with controls. On the other hand, small intestinal length and weight were increased in IL-10 gene-deficient mice compared with controls, and these were reduced by 3-aminobenzamide treatment. There was no difference in large intestinal length among the three groups; however, large intestinal weight was significantly increased in IL-10 gene-deficient mice. Again, 3-aminobenzamide treatment reduced colonic weight in the IL-10 gene-deficient mice. Wet weights of heart, lung, liver, and kidney did not differ between groups (data not shown).

                              
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Table 2.   Morphological characteristics of 10-wk-old control mice, IL-10 gene-deficient mice, and IL-10 gene-deficient mice receiving 3-aminobenzamide for 14 days

At 10 wk of age, IL-10 gene-deficient mice displayed a patchy distribution of transmural acute and chronic inflammation, extensive mucosal ulceration, and epithelial hyperplasia (Fig. 1; Table 3). In contrast, those mice treated with 3-aminobenzamide demonstrated significantly improved mucosal architecture with no mucosal ulceration or neutrophilic infiltrate (Fig. 1; Table 3). Approximately 25% of the 3-aminobenzamide-treated mice exhibited minor epithelial hyperplasia and/or the presence of a mononuclear infiltrate in the lamina propria. Those mice receiving the structural analog 3-aminobenzoic acid demonstrated no significant improvement in histological score (Table 3).


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Fig. 1.   Representative photomicrographs from colons of 10-wk-old mice. A: control mouse colon (magnification = ×100). B: interleukin (IL)-10 gene-deficient mouse colon. Note the mucosal ulceration, transmural inflammation, and formation of crypt abscesses (magnification = ×40; inset magnification = ×400). C: colon from IL-10 gene-deficient mouse receiving 3-aminobenzamide for 14 days (magnification = ×100).


                              
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Table 3.   Histological assessment of colons from control mice, IL-10 gene-deficient mice, and IL-10 gene-deficient mice receiving either 3-aminobenzamide or 3-aminobenzoic acid

Cytokine measurement. Previous studies have shown that immune cells found in colonic tissue from IL-10 gene-deficient mice spontaneously produce higher levels of proinflammatory cytokines compared with immune cells from wild-type mice (5). To determine whether the improvement in histological score induced by 3-aminobenzamide treatment was accompanied by an alteration in cytokine production, TNF-alpha and IFN-gamma secretion were measured. As previously shown (28), colons from IL-10 gene-deficient mice spontaneously produced higher levels of TNF-alpha and IFN-gamma compared with control mice (Fig. 2), likely because of the large influx of lymphocytes into the lamina propria. In addition, IL-10 gene-deficient mice also produced higher levels of TNF-alpha and IFN-gamma in response to LPS stimulation compared with control mice. In correlation with the attenuation of inflammation and reduction of lymphocytic infiltration, spontaneous and LPS-stimulated colonic secretion of both TNF-alpha and IFN-gamma were significantly reduced in those mice receiving 3-aminobenzamide for 2 wk (Fig. 2). Furthermore, in control and 3-aminobenzamide-treated mice, there was no significant difference in spontaneous and LPS-stimulated cytokine release. In contrast, in IL-10 gene-deficient mice, LPS-stimulation resulted in a significant increase in proinflammatory cytokine release, suggesting the presence of monocytes within the lamina propria (Fig. 2).


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Fig. 2.   Secretion of tumor necrosis factor (TNF)-alpha (A) and interferon (IFN)-gamma (B) by colonic epithelium from control mice, IL-10 gene-deficient mice, and IL-10 gene-deficient mice receiving 3-aminobenzamide (3-AB) for 14 days. Compared with control mice, IL-10 gene-deficient mice produced significantly greater amounts of TNF-alpha and IFN-gamma both under basal conditions and with lipopolysaccharide (LPS) stimulation. IL-10 gene-deficient mice receiving 3-aminobenzamide demonstrated significant reductions in secretion of proinflammatory cytokines under both basal and LPS-stimulated conditions. * P < 0.05 compared with control mice. + P < 0.05 compared with basal period.

NOS assessment. NO is produced by two distinct isoforms in epithelial tissue. Constitutive NOS (cNOS) is a calcium-dependent constitutive enzyme, whereas iNOS is a calcium-independent isoform whose expression is induced in response to various insults, including TNF-alpha (34). iNOS activity was assessed enzymatically, and protein levels were assessed by Western blotting. Colonic tissue from IL-10 gene-deficient mice demonstrated high levels of iNOS activity (Fig. 3A) and iNOS protein expression (Fig. 3B). Both iNOS activity and protein levels were significantly reduced after 2 wk of 3-aminobenzamide treatment. This reduction of iNOS activity likely reflects a decrease in iNOS-expressing cells in the lamina propria, since histological assessment revealed a significant decrease in lymphocytic infiltration after 3-aminobenzamide treatment. On the other hand, cNOS activity was not different among the three groups (data not shown).


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Fig. 3.   Inducible nitric oxide synthase (iNOS) enzymatic activity (A) and protein levels (B) in colonic mucosa. Lane 1 represents control mice; lane 2 represents IL-10 gene-deficient mice; lane 3 represents IL-10 gene-deficient mice receiving 3-aminobenzamide for 14 days. iNOS activity and protein levels were significantly increased in IL-10 gene-deficient mice compared with control mice. Those mice receiving 3-aminobenzamide demonstrated a significant reduction in iNOS activity and protein levels. * P < 0.01 compared with control mice. + P < 0.01 compared with IL-10 gene-deficient mice.

Nitrotyrosine formation. The interaction of proinflammatory cytokines with specific receptors on the plasma membranes of phagocytes can lead to the activation of the plasma membrane-associated flavoprotein NADPH oxidase and the subsequent release of large amounts of SO and hydrogen peroxide. SO can then react with NO to form the very potent oxidant peroxynitrite (18). IL-10 gene-deficient mice demonstrated increased levels of TNF-alpha and IFN-gamma and iNOS activity. This, coupled with the very large influx of leukocytes into the lamina propria (see Table 3), could result in high concentrations of reactive oxygen and nitrogen species (9). The formation of nitrotyrosine adducts in tissue serves as an indicator of the presence of reactive nitrogen species (17). Assessment of total levels of nitrotyrosine by Western blotting revealed evidence of increased levels of nitrosylated proteins in IL-10 gene-deficient mice, as exemplified by the appearance of a new nitrosylated protein at ~70 kDa and an increase in the levels of a protein migrating at ~25 kDa (Fig. 4). In conjunction with the reduced levels of TNF-alpha and iNOS activity, 3-aminobenzamide-treated IL-10 gene-deficient mice also had significantly reduced levels of the 70-kDa nitrotyrosine band. The identity of these proteins is unknown, although previous studies have also described a low-molecular-weight nitrosylated protein that migrates close to 25 kDa (7).


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Fig. 4.   Representative antinitrotyrosine blot of colonic mucosa from control mice, IL-10 gene-deficient (-/-) mice, and IL-10 gene-deficient mice receiving 3-aminobenzamide (3-AB). Approximate molecular masses of immunoreactive bands are noted at right. IL-10 gene-deficient mice receiving 3-aminobenzamide demonstrated reduced levels of nitrotyrosine at 70 kDa compared with IL-10 gene-deficient mice. Control mice showed no nitrotyrosine reactivity at 70 kDa.

PARP activity. Reactive oxygen and nitrogen species are able to indiscriminately attack DNA, resulting in DNA strand breakage (47). This DNA damage subsequently activates the nuclear enzyme PARP (43). Thus, to determine whether the presence of increased levels of nitrotyrosine in IL-10 gene-deficient mice was associated with enhanced levels of PARP activity, mucosa was scraped and PARP activity was measured. As seen in Fig. 5, PARP activity was enhanced approximately fourfold in 10-wk-old IL-10 gene-deficient mice compared with controls. 3-Aminobenzamide treatment for 14 days completely normalized PARP activity in IL-10 gene-deficient mice (Fig. 5).


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Fig. 5.   Colonic poly(ADP-ribose) polymerase (PARP) activity was significantly increased in IL-10 gene-deficient mice compared with control mice and IL-10 gene-deficient mice receiving 3-aminobenzamide for 14 days. * P < 0.01 compared with control mice. + P < 0.01 compared with IL-10 gene-deficient mice.

Colonic permeability. In vitro studies have demonstrated that increased paracellular permeability can occur as a result of exposure to exogenous NO or peroxynitrite (19, 43). Moreover, inhibition of PARP activity prevents the increase in paracellular permeability induced by peroxynitrite (19). To determine whether inhibition of PARP activity in vivo would have a similar effect in a chronic model of colitis, colonic permeability was measured in mice using a single-pass perfusion technique. At 10 wk of age, IL-10 gene-deficient mice had significantly increased colonic permeability and decreased water absorption compared with age-matched control mice (Fig. 6). On the other hand, IL-10 gene-deficient mice treated with 3-aminobenzamide for 14 days displayed normal colonic permeability and water absorption (Fig. 6).


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Fig. 6.   Colonic permeability as assessed by mannitol clearance (A) and water absorption (B) in 10-wk-old mice. IL-10 gene-deficient mice had significantly greater colonic permeability compared with age-matched controls. 3-Aminobenzamide treatment for 14 days normalized both mannitol clearance and water absorption. * P < 0.05 compared with controls. + P < 0.05 compared with IL-10 gene-deficient mice.

Metabolite levels. Levels of ATP, ADP, AMP, and lactate were measured in whole colon homogenates to determine whether the increase in colonic permeability observed in IL-10 gene-deficient mice was associated with altered energy status. Compared with control mice, IL-10 gene-deficient mice had significant alterations in colonic cellular energy status, demonstrating increases in cellular ADP and lactate (P < 0.05) and suggesting an apparent shunting of energy production from oxidative phosphorylation to anaerobic glycolysis (Table 4). Interestingly, total levels of cellular ATP were increased in colons from IL-10 gene-deficient mice compared with control colons. Inhibition of PARP activity with 3-aminobenzamide resulted in decreases in cellular ADP and lactate and a further increase in cellular ATP levels and cellular energy charge (Table 4). Thus disease in IL-10 gene-deficient mice was not associated with decreased total energy charge but was associated with high levels of lactate. This would suggest that lactate acidosis and not reduced levels of ATP may be involved in alterations of intestinal permeability.

                              
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Table 4.   Levels of metabolites in colons from 10-wk-old control mice, IL-10 gene-deficient mice, and IL-10 gene-deficient mice receiving 3-aminobenzamide for 14 days

Acute Treatment of IL-10 Gene-Deficient Mice

There is evidence that inhibition of PARP reduces the recruitment of neutrophils into intestinal tissue by inhibiting the expression of adhesion molecules (48-50). In addition, inhibition of PARP activity can limit the release of proinflammatory cytokines from macrophages (21). Thus the improvement of inflammation in IL-10 gene-deficient mice treated for 14 days may have been initiated either by a 3-aminobenzamide-induced inhibition of neutrophil infiltration into the lamina propria or a 3-aminobenzamide-induced downregulation of cytokine release. Alternatively, if epithelial permeability was corrected first by a 3-aminobenzamide effect on cellular energetic status, this would reduce the level of antigenic materials entering the lamina propria and thus remove the antigenic trigger that may stimulate neutrophilic influx and cytokine release.

We examined the effects of a single injection of 3-aminobenzamide in 3-wk-old IL-10 gene-deficient mice to determine a time course of 3-aminobenzamide-induced changes in cellular energy status, permeability, and proinflammatory cytokine release. At 3 wk of age, IL-10 gene-deficient mice exhibit an increase in colonic permeability, with no histological evidence of neutrophilic infiltration or ulceration (28). However, mice at this age do demonstrate increased TNF-alpha and IFN-gamma secretion. As seen in Fig. 7C, a single injection of 3-aminobenzamide produced a significant reduction in colonic mannitol clearance in 3-wk-old IL-10 gene-deficient mice by 2 h. Coinciding with the improvement in permeability were decreases in cellular lactate levels (Table 5). Total cellular ATP levels were not different among controls, IL-10 gene-deficient mice, and IL-10 gene-deficient mice receiving 3-aminobenzamide either at 2 or 4 h after injection. In contrast to the rapid effects of 3-aminobenzamide treatment on cellular metabolic function and permeability, TNF-alpha (Fig. 7A) and IFN-gamma (Fig. 7B) secretion were not significantly reduced until 4 h after 3-aminobenzamide injection. This would suggest that inhibition of PARP activity with 3-aminobenzamide has a direct effect on cellular metabolic function independent of and before a suppression of proinflammatory cytokine release and neutrophilic influx.


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Fig. 7.   Effects of acute treatment with 3-aminobenzamide on secretion of TNF-alpha (A) and IFN-gamma (B) and on colonic mannitol clearance (C) in 3-wk-old IL-10 gene-deficient mice. Two hours after a single injection of 3-aminobenzamide (20 mg/kg), mannitol clearance was significantly reduced. Four hours after 3-aminobenzamide injection, colonic TNF-alpha and IFN-gamma secretion were significantly reduced. * P < 0.05 compared with control mice. + P < 0.05 compared with IL-10 gene-deficient mice.


                              
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Table 5.   Levels of metabolites in colons from 3-wk-old control mice, IL-10 gene-deficient mice, and IL-10 gene-deficient mice receiving a single injection of 3-aminobenzamide


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrate for the first time that established colonic inflammatory disease can be effectively treated by pharmacological inhibition of PARP activity. In addition, we have demonstrated a new mechanism for the beneficial effects of PARP inhibition, that being a normalization of cellular metabolic function and intestinal permeability.

Colitis in the IL-10 gene-deficient mouse develops shortly after weaning at ~4 wk of age (27). By 8 wk of age, colitis is well established and is characterized by patchy mucosal ulceration, extensive neutrophilic infiltration into the lamina propria, and epithelial hyperplasia (5). Disease in IL-10 gene-deficient mice is initiated by CD4+ T cells and results in an IL-12- and IFN-gamma -directed excessive generation and activation of Th1 cells (5). Similar to what is seen in patients with Crohn's disease, colonic inflammation in this model is associated with increased permeability, high levels of mucosal IFN-gamma and TNF-alpha , and increased NO production (28). The cause of the increased permeability in IL-10 gene-deficient mice exhibiting colonic inflammation may be due to increased levels of peroxynitrite, a reaction product of NO and the SO anion and a common effector of tissue injury during inflammation (4, 47). In this study, we found evidence for elevated levels of mucosal nitrotyrosine in colonic tissue from adult IL-10 gene-deficient mice, which is suggestive of nitrosative stress and a reaction between peroxynitrite and tyrosine residues (4). Indeed, NO has been shown in vitro to mediate IFN-gamma -induced increases in permeability (45), and, furthermore, Kennedy et al. (19) have reported that nitrosative stress increases epithelial permeability in intestinal monolayers via DNA damage and a subsequent activation of the enzyme PARP. In conjunction with the increased levels of nitrotyrosine, we also observed an elevation of colonic PARP activity.

The mechanism behind a PARP-induced increase in epithelial permeability has been suggested to be a reduction in cellular ATP levels and a resultant breakdown of tight junctional integrity (43). Indeed, this has clearly been shown to occur in intestinal monolayers in vitro through a PARP-induced depletion of cellular NAD+ and a subsequent suppression of ATP synthesis (19). However, we were unable to demonstrate a similar reduction in tissue ATP levels in vivo. In fact, colonic homogenates from IL-10 gene-deficient mice demonstrated higher levels of cellular ATP compared with control mice. This may be due to the fact that we examined whole tissue ATP levels, which reflect ATP levels of numerous cell types. In addition, diseased mice have a high number of recruited immune cells present in the lamina propria. However, we did observe significant alterations in ADP and lactate levels in colonic tissue from both 3-wk-old and 10-wk-old IL-10 gene-deficient mice, suggesting an increased demand for ATP and a shunting of energy production from oxidative phosphorylation to anaerobic glycolysis both before the development of histological inflammation (3 wk old) and in the presence of active inflammation (10 wk old). Although the reason for this shunting is unknown, it has previously been shown that an active inflammatory state results in elevated energy expenditure and enhanced protein catabolism (20, 24). Indeed, IL-10 gene-deficient mice exhibited a net weight loss over the 14-day treatment period, despite consuming the same amount of food as did control mice. The length of the small intestine was also significantly increased in IL-10 gene-deficient mice, likely as an adaptation to this hypermetabolic state. Oxidative damage to mitochondria could also cause shunting of energy production toward anaerobic glycolysis. IL-10 gene-deficient mice exhibit high levels of proinflammatory cytokines and nitrotyrosine; thus a loss of function of mitochondria in these mice is very possible. Furthermore, activation of PARP itself can inhibit mitochondrial activity. Indeed, we found that inhibition of PARP rapidly restored lactate levels and colonic permeability to normal in both 3-wk-old and 10-wk-old mice, suggesting the involvement of PARP activity. Activated PARP consumes the ADP ribose moiety of NAD+ to synthesize ADP-ribose polymers on a variety of nuclear target proteins, including DNA polymerase I, histones, and PARP itself in a futile cycle that can deplete cellular NAD+ (22). This PARP-induced depletion of cellular NAD+ can inhibit mitochondrial respiration and result in enhanced levels of anaerobic glycolysis (22). Interestingly, the increased levels of cellular lactate suggest the presence of cellular acidosis, and it may be the acidosis that is altering intestinal permeability in vivo, as has been seen in endotoxin-induced mucosal acidosis in pigs (39). Indeed, low extracellular pH has been shown to increase epithelial permeability in Caco-2BBe cells (31) and potentiate NO-induced increases in intestinal permeability in vitro (44). Further studies are necessary to determine exactly which cells (epithelia, fibroblasts, immune) are exhibiting alterations in metabolic function in the colons of IL-10 mice and the role of PARP activity in these alterations.

Previous studies have clearly shown that pharmacological inhibition of PARP activity is beneficial in various acute models of inflammation, such as stroke (43), endotoxic shock (42), and ischemia-reperfusion injury (49). In addition, Zingarelli and co-workers (50) demonstrated that PARP-deficient animals are protected from the lethal effects of trinitrobenzene sulfonic acid (TNBS) colitis. However, unlike the IL-10 gene-deficient mouse model, TNBS colitis is not spontaneous and is ultimately resolved in time (33). In contrast, in this study we demonstrate that PARP inhibition is beneficial in a chronic model of colitis and that treatment of established colitis by pharmacological inhibition of PARP dramatically improves barrier and metabolic function and, subsequently, inflammation.

There are several mechanisms by which long-term inhibition of PARP activity may lead to resolution of colitis. Zingarelli et al. (49, 50) suggested that inhibition of PARP activation reduces intracellular adhesion molecule-1 and P-selectin expression, leading to an inhibition of neutrophil recruitment and decreased oxidant generation in the lamina propria. Our data showing that neutrophil infiltration and nitrotyrosine formation are significantly reduced after 14 days of treatment with 3-aminobenzamide support this hypothesis. However, in our study we show that intestinal permeability is enhanced in 3-wk-old mice in the absence of any neutrophilic infiltration. Furthermore, a single injection of 3-aminobenzamide can restore metabolic alterations and intestinal permeability to normal within 2 h, arguing against the notion that the primary beneficial effects of PARP inhibition are due to changes in adhesion molecule expression.

In addition to NO and peroxynitrite, proinflammatory cytokines such as IFN-gamma and TNF-alpha also increase permeability in epithelial monolayers, albeit possibly through a NO-mediated mechanism (26, 36). Colonic secretion of both IFN-gamma and TNF-alpha are increased in IL-10 gene-deficient mice before the development of histological inflammation and neutrophilic infiltration (28), suggesting the presence of activated T cells and macrophages. In macrophages, an ADP-ribosylation reaction mediated by PARP appears to contribute to the activation of nuclear factor (NF)-kappa B and precedes the upregulation of iNOS activity and release of proinflammatory cytokines (20). Inhibition of PARP prevents the release of these cytokines and, indeed, attenuates NF-kappa B activation in macrophages (21). Thus treatment of mice with 3-aminobenzamide in vivo may downregulate cytokine release from macrophages and, in this context, improve permeability. However, the acute studies clearly demonstrate that 2 h after a single injection of 3-aminobenzamide, colonic permeability is significantly reduced, whereas attenuation of TNF-alpha and IFN-gamma secretion did not occur until 4 h after 3-aminobenzamide injection. Nonetheless, the inhibition of proinflammatory cytokine release from macrophages likely contributes to the beneficial effects of PARP inhibition.

Another potential mechanism by which PARP inhibition may act to improve barrier function and suppress inflammation is the prevention of necrosis by colonic epithelial cells following oxidant-mediated injury (46). It has been suggested that a lack of sufficient ATP results in a damaged cell undergoing necrosis, a proinflammatory event, rather than apoptosis (46). However, our acute studies demonstrating that PARP inhibition attenuates the permeability defect within 2 h after injection would suggest that prevention of necrosis is not the initiating factor in the suppression of inflammation, but, again, this may contribute to the beneficial effects of PARP inhibition.

Together, these data support a role for PARP in the alteration of cellular metabolic function and intestinal permeability in the IL-10 gene-deficient mouse. Although it is difficult to determine from this study the relative contribution of various PARP activation pathways (i.e., enterocyte, endothelial cell, immune cell) to mucosal inflammation, this work does highlight a role for PARP and poly ADP-ribosylation mechanisms in the perpetuation of chronic inflammation. Indeed, this study demonstrates that inhibition of PARP results in a dramatic improvement of mucosal function in a chronic model of intestinal inflammation. This likely occurs through multiple mechanisms, including a rapid restoration of epithelial barrier integrity through a correction of cellular metabolic function. Therefore, this study supports PARP inhibition as a therapeutic tool in the treatment of inflammatory bowel disease.


    ACKNOWLEDGEMENTS

This work was supported by the Alberta Heritage Foundation for Medical Research, the Crohn's and Colitis Foundation of Canada, and Schering Canada, which supported the IL-10 gene-deficient mouse colony.


    FOOTNOTES

Address for reprint requests and other correspondence: K. L. Madsen, Univ. of Alberta, 536 Newton Bldg., Edmonton, Alberta T6G 2C2, Canada (E-mail: karen.madsen{at}ualberta.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 17 April 2000; accepted in final form 13 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Atkinson, DE. The energy charge of the adenylate pool as a regulatory parameter. Biochemistry 7: 4030-4034, 1968[ISI][Medline].

2.   Ballard, ST, Hunter JH, and Taylor AE. Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium. Annu Rev Nutr 15: 35-55, 1995[ISI][Medline].

3.   Banasik, M, Komura H, Shimoyama M, and Ueda K. Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl) transferase. J Biol Chem 267: 1569-1575, 1992[Abstract/Free Full Text].

4.   Beckman, JS. Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol 9: 836-844, 1996[ISI][Medline].

5.   Berg, DJ, Davidson N, Kuhn R, Muller W, Menon S, Holland G, Thompson-Snipes L, Leach MW, and Rennick D. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses. J Clin Invest 98: 1010-1020, 1996[Abstract/Free Full Text].

6.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

7.   Briviba, K, Klotz L, and Sies H. Defenses against peroxynitrite. Methods Enzymol 301: 301-311, 1999[ISI][Medline].

8.   Casellas, F, Aguade S, Soriano B, Accarino A, Molero J, and Guarner L. Intestinal permeability to 99mTc-diethylenetriaminopentaacetic acid in inflammatory bowel disease. Am J Gastroenterol 81: 767-770, 1986[ISI][Medline].

9.   Eiserich, JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B, and van der Vliet A. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391: 393-397, 1998[ISI][Medline].

10.   Eliasson, MJ, Sampei K, Mandir AS, Hurn PD, Traystman RJ, Bao J, Pieper A, Wang ZQ, Dawson TM, Snyder SH, and Dawson VL. Poly(ADP-ribose) polymerase gene disruption renders mice resistance to cerebral ischemia. Nat Med 3: 1089-1095, 1997[ISI][Medline].

11.   Gardiner, KR, Anderson NH, Rowlands BJ, and Barbul A. Colitis and colonic barrier dysfunction. Gut 37: 530-535, 1995[Abstract].

12.   Go, LL, Healey PJ, Watkins SC, Simmons RL, and Rowe MI. The effect of endotoxin on intestinal mucosal permeability to bacteria in vitro. Arch Surg 130: 53-58, 1995[Abstract].

13.   Grisham, MB. Oxidants and free radicals in inflammatory bowel disease. Lancet 344: 859-861, 1994[ISI][Medline].

14.   Hecht, G, Koutsouris A, Pothoulakis C, LaMont JT, and Madara JL. Clostridium difficile toxin B disrupts the barrier function of T84 monolayers. Gastroenterology 102: 416-423, 1992[ISI][Medline].

15.   Hecht, G, Pothoulakis C, LaMont JT, and Madara JL. Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers. J Clin Invest 82: 1516-1524, 1998.

16.   Hollander, D, Vadheim CM, Brettholz E, Petersen GM, Delahunty T, and Rotter JL. Increased intestinal permeability in patients with Crohn's disease and their relatives. A possible etiologic factor. Ann Intern Med 105: 883-885, 1986[ISI][Medline].

17.   Ischiropoulos, H, Zhu L, Chen J, Tsai M, Martin JC, Smith CD, and Beckman JS. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys 298: 431-437, 1992[ISI][Medline].

18.   Jourd'heuil, D, Morise Z, Conner EM, and Grisham MB. Oxidants, transcription factors, and intestinal inflammation. J Clin Gastroenterol 25, Suppl 1: S61-S72, 1997[ISI][Medline].

19.   Kennedy, M, Denenberg AC, Szabo C, and Salzman AL. Poly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells. Gastroenterology 114: 510-518, 1998[ISI][Medline].

20.   Lennie, TA, McCarthy DO, and Keesey RE. Body energy status and the metabolic response to acute inflammation. Am J Physiol Regulatory Integrative Comp Physiol 269: R1024-R1031, 1995[Abstract/Free Full Text].

21.   Le Page, C, Sanceau J, Drapier JC, and Wietzerbin J. Inhibitors of ADP-ribosylation impair inducible nitric oxide synthase gene transcription through inhibition of NF kappa B activation. Biochem Biophys Res Commun 243: 451-457, 1998[ISI][Medline].

22.   Le Rhun, Y, Kirkland JB, and Shah GM. Cellular responses to DNA damage in the absence of poly(ADP-ribose) polymerase. Biochem Biophys Res Commun 245: 1-10, 1998[ISI][Medline].

23.   Lindmark, T, Kimura Y, and Artursson P. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J Pharmacol Exp Ther 284: 362-369, 1998[Abstract/Free Full Text].

24.   Ling, PR, Bistrian BR, Mendez B, and Istfan NW. Effects of systemic infusions of endotoxin, tumor necrosis factor, and interleukin-1 on glucose metabolism in the rat: relationship to endogenous glucose production and peripheral tissue glucose uptake. Metabolism 43: 279-284, 1994[ISI][Medline].

25.   Lowry, OH, and Passonneau JV. A flexible system of enzymatic analysis. New York: Academic, 1972.

26.   Madara, JL, and Stafford J. Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers. J Clin Invest 83: 724-727, 1989[ISI][Medline].

27.   Madsen, KL, Doyle JS, Jewell LD, Tavernini MM, and Fedorak RN. Lactobacillus species prevents colitis in interleukin-10 gene-deficient mice. Gastroenterology 116: 1-9, 1999.

28.   Madsen, KL, Malfair D, Gray DJ, Doyle JS, Jewell LD, and Fedorak RN. Interleukin-10 gene-deficient mice develop a primary intestinal permeability defect in response to enteric microflora. Inflamm Bowel Dis 5: 262-270, 1999[ISI][Medline].

29.   Madsen, KL, Yanchar NL, Sigalet DL, Reigel T, and Fedorak RN. FK506 increases permeability in rat intestine by inhibiting mitochondrial function. Gastroenterology 109: 107-114, 1995[ISI][Medline].

30.   Mandel, LJ, Bacallao R, and Zampighi G. Uncoupling of the molecular "fence" and paracellular "gate" functions in epithelial tight junctions. Nature 361: 552-555, 1993[ISI][Medline].

31.   Menconi, MJ, Salzman AL, Unno N, Ezzel RM, Casey DM, Tsuji Y, and Fink MP. Acidosis induces hyperpermeability in Caco-2BBe cultured intestinal epithelial monolayers. Am J Physiol Gastrointest Liver Physiol 272: G1007-G1021, 1997[Abstract/Free Full Text].

32.   Miller, DL, and Schedl HP. Total recovery studies of nonabsorbable indicators in the rat small intestine. Gastroenterology 58: 40-46, 1970[ISI][Medline].

33.   Morris, GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, and Wallace JL. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795-803, 1989[ISI][Medline].

34.   Nathan, C, and Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915-918, 1994[ISI][Medline].

35.   Peeters, M, Geypens B, Claus D, Nevens H, Ghoos Y, Verbeke G, Baert F, Vermeire S, Vlietinck R, and Rutgeerts P. Clustering of increased small intestinal permeability in families with Crohn's disease. Gastroenterology 113: 802-807, 1997[ISI][Medline].

36.   Rodriguez, P, Heyman M, Candalh C, Blaton MA, and Bouchaud C. Tumour necrosis factor-alpha induces morphological and functional alterations of intestinal HT29 cl.19A cell monolayers. Cytokine 7: 441-448, 1995[ISI][Medline].

37.   Sadowski, DC, and Meddings JB. Luminal nutrients alter tight-junction permeability in the rat jejunum: an in vivo perfusion model. Can J Physiol Pharmacol 71: 835-839, 1993[ISI][Medline].

38.   Salzman, A, Menconi MJ, Unno N, Ezzell RM, Casey DM, Gonzalez PK, and Fink MP. Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers. Am J Physiol Gastrointest Liver Physiol 268: G361-G373, 1995[Abstract/Free Full Text].

39.   Salzman, AL, Wang H, Wollert PS, VanderMeer TJ, Compton CC, Denenberg AG, and Fink MP. Endotoxin-induced ileal mucosal hyperpermeability in pigs: role of tissue acidosis. Am J Physiol Gastrointest Liver Physiol 266: G633-G646, 1994[Abstract/Free Full Text].

40.   Saverymuttu, SH, Camilleri M, Rees H, Lavender JP, Hodgson HJ, and Chadwick VS. Indium 111-granulocyte scanning in the assessment of disease extent and disease activity in inflammatory bowel disease. A comparison with colonoscopy, histology, and fecal indium 111-granulocyte excretion. Gastroenterology 90: 1121-1128, 1986[ISI][Medline].

41.   Singer, II, Kawka DW, Scott S, Weidner JR, Mumford RA, Riehl TE, and Stenson WF. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 111: 871-885, 1996[ISI][Medline].

42.   Szabo, C, Cuzzocrea S, Zingarelli B, O'Connor M, and Salzman AL. Endothelial dysfunction in a rat model of endotoxic shock: importance of the activation of poly (ADP-ribose) synthetase by peroxynitrite. J Clin Invest 100: 723-735, 1997[Abstract/Free Full Text].

43.   Szabo, C, and Dawson VL. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion. Trends Pharmacol Sci 19: 287-298, 1998[ISI][Medline].

44.   Unno, N, Menconi MJ, Salzman AL, Smith M, Hagen S, Ge Y, Ezzell RM, and Fink MP. Hyperpermeability and ATP depletion induced by chronic hypoxia or glycolytic inhibition in Caco-2BBe monolayers. Am J Physiol Gastrointest Liver Physiol 270: G1010-G1021, 1996[Abstract/Free Full Text].

45.   Unno, N, Menconi MJ, Smith M, and Fink MP. Nitric oxide mediates interferon-gamma -induced hyperpermeability in cultured human intestinal epithelial monolayers. Crit Care Med 23: 1170-1176, 1995[ISI][Medline].

46.   Watson, AJ. Manipulation of cell death---the development of novel strategies for the treatment of gastrointestinal disease. Aliment Pharmacol Ther 9: 215-226, 1995[ISI][Medline].

47.   Wiseman, H, and Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 313: 17-29, 1996[ISI][Medline].

48.   Zingarelli, B, Cuzzocrea S, Zsengeller Z, Salzman AL, and Szabo C. Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase. Cardiovasc Res 36: 205-215, 1997[ISI][Medline].

49.   Zingarelli, B, Salzman AL, and Szabo C. Genetic disruption of poly (ADP-ribose) synthetase inhibits the expression of P-selectin and intercellular adhesion molecule-1 in myocardial ischemia/reperfusion injury. Circ Res 83: 85-94, 1998[Abstract/Free Full Text].

50.   Zingarelli, B, Szabo C, and Salzman AL. Blockade of Poly(ADP-ribose) synthetase inhibits neutrophil recruitment, oxidant generation, and mucosal injury in murine colitis. Gastroenterology 116: 335-345, 1999[ISI][Medline].


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