INTRODUCTION
Acute mesenteric ischemia followed by reperfusion (ischemia/reperfusion) may be caused by vascular disease, hemorrhage, or trauma, and is associated with a high morbidity and mortality despite modern intensive care therapy (1). Potential mechanisms causing the development and progression of ischemia/reperfusion injury include the adhesion and activation of polymorphonuclear neutrophils, the release of proinflammatory cytokines, as well as the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (2,3). ROS, produced during ischemia/reperfusion, have also been implicated in a number of signal transduction pathways (4). Among the important radicals produced during ischemia/reperfusion is the bioregulatory molecule nitric oxide (NO), generated catalytically by three enzymes collectively termed NO synthases (NOS) (5,6). Recent work has suggested that NO produced by the endothelial, constitutive NOS (ecNOS) may be an important endogenous protective molecule that limits the tissue injury caused by ischemia/reperfusion of the small bowel (7,8). For instance, inhibitors of endogenous NO production greatly exacerbate the increase in epithelial permeability and the cardiovascular dysfunction caused by ischemia/reperfusion of the intestine (9), while NO donors prevent the early rise in epithelial permeability (10) and the associated tissue dysfunction (9,11). Intra-arterial infusion of L-arginine (the substrate for NOS) at the onset of reperfusion protects the intestine against postischemic injury, presumably by enhancing the formation of NO (2). Excess NO production has been attributed to a second NOS that is not present under normal conditions, but can be induced within 2 h, reaching significant levels by 4 to 6 h in various cells (5,6). The induction of inducible NOS (iNOS) has been implicated in the pathogenesis of ischemia/reperfusion injury, shock, and inflammation. NO can have both direct effects on cell signalling, as well as indirect actions mediated by the reaction products formed when NO interacts with other molecules such as oxygen or superoxide to form RNS (12). In this study, we hypothesize that enhanced NO formation by iNOS may contribute to tissue injury and proinflammatory signalling in ischemia/reperfusion injury of the mesentery. Therefore. studies involving splanchnic ischemia/reperfusion were performed in mice treated with the novel, selective, and potent inhibitor of iNOS, GW274150 [(S)-2-amino-(1-iminoethylamino)-5-thioheptanoic acid] (13) and in mice genetically deficient in the gene for iNOS. GW274150 is a novel NOS inhibitor (sulfur-substituted acetamine amino acid) that acts in competition with L-arginine and has a very high degree of selectivity for iNOS when compared with either eNOS (>300-fold) or nNOS (>100-fold). Like with 1400W, the inhibition of iNOS activity caused by GW274150 is NADPH dependent and develops very slowly, but is rapidly reversible (14,15).
MATERIALS AND METHODS
Animals
Homozygous iNOS−/− and iNOS+/+ (wild-type C57B1/6 × 129/Sv), male mice (20–25 g, kindly supplied by Fons A.J. Van de Loo, Department of Rheumatology, University Hospital Nijmegen, Nijmegen, The Netherlands) were used to assess the role of iNOS in the pathogenesis of splanchnic ischemia/reperfusion shock. A neocassette using homologous recombination as previously described (16) replaced the first four exons of the NOS2 gene. All animals were allowed access to food and water ad libitum. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986)
Splanchnic ischemia/reperfusion
Animals were anesthetized with sodium thiopentone (4 mg/mL, 10 μL/g weight of mouse) and placed in a supine position. After a midline laparotomy, the celiac trunk and the superior mesenteric artery were isolated close to their aortic origins. During this procedure, the intestinal tract was maintained at 37°C by placing it between gauze pads soaked with warmed saline solution. In pilot studies, splanchnic ischemia (SAO shock) was produced by clamping both arteries for 45 min, as previously described (3). After this period of occlusion, reperfusion was induced by removing the clamps. In a second study, we used a less severe model of mesenteric infarction (SMO), obtained by only occlusion of the superior mesenteric artery only for 45 min followed by reperfusion. Survival was monitored for 4 h in both models of gut ischemia. All groups of mice were sacrificed 4 h after onset of reperfusion, and ileum tissues were rapidly removed for quantification of bowel injury and immunohistochemical and biochemical analyses. Blood samples were obtained through cardiac puncture. An additonal group of mice underwent the above surgical procedure with the exception of the occlusion and reperfusion of the splanchnic arteries, and this group served as a sham-control group. In a separate set of experiments, SAO or SMO shock was induced in iNOS wild-type (WT) treated with GW274150 (5 mg/kg i.p.) 30 min before reperfusion.
Measurement of NOS activity
The calcium-independent conversion of L-arginine to L-citrulline in the intestine homogenates (obtained after 4 h of reperfusion) served as an indicator of iNOS activity (15). Intestine was homogenated into a homogenation buffer composed of 50 mM Tris-HCl, 0.1 mM EDTA, and 1 mM phenylmethylsufonyl fluoride (pH 7.4), and was homogenized on ice using a tissue homogenizer. Conversion of [3H]L-arginine to [3H]L-citrulline was measured in the homogenates as described (12,17). Briefly, homogenate (30 μL) was incubated in the presence of [3H]L-arginine ( 10 μM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 μM), and calcium (2 mM) for 30 min at 22°C. Reactions were stopped by dilution with 0.5 mL of ice-cold HEPES buffer (pH 5.5) containing EGTA (2 mM) and EDTA (2 mM). Experiments performed in the absence of NADPH determined the extent of [3H]L-citrulline formation independent of specific NOS activity. Experiments in the presence of NADH, without calcium and EGTA (5 mM), determined the calcium-independent (i.e., induced) NOS activity. Reaction mixtures were applied to Dowex 50W (Na+ form) columns, and the eluted [3H]L-citrulline activity was measured by a scintillation counter (Wallac, Gaithersburg, MD).
Immunohistochemical localization of P-selectin, intracellular adhesion molecule-1 (ICAM-1), poly(ADP-ribosylated) (PAR) proteins and nitrotyrosine
Immunohistochemical staining was performed on 7-μm-thick sections of unfixed mice ileum. Sections were cut in with a Slee and London cryostat at −30°C, were transferred onto clean glass slides, and were dried overnight at room temperature. Sections were permeabilized with acetone at −20°C for 10 min and were rehydrated in phosphate-buffered saline (PBS, 150 mM NaCl and 20 mM sodium phosphate, pH 7.2) at room temperature for 45 min. Sections were incubated overnight with one of the following: (1) rabbit anti-human polyclonal antibody directed at P-selectin (CD62P; 1:500 in PBS, v/v; DBA, Milan, Italy), (2) mouse anti-rat antibody directed at ICAM-1 (CD54; 1:500 in PBS, v/v; DBA), (3) anti-poly(ADP-ribose) monoclonal antibody (1:500 in PBS, v/v; DBA), or (4) anti-nitrotyrosine antibody (1:500 in PBS, v/v; DBA). Sections were washed with PBS and incubated with secondary antibody for 2 h at room temperature. Specific labelling was detected using an avidin-biotin peroxidase complex.
Histopathological analysis and damage score
Ileum tissue was fixed in PBS-buffered 10% (w/v) formaldehyde solution at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (7-μm thickness) were deparaffinized with xylene, stained with Alcyan Bleu-PASS, and studied using light microscopy (Dialux 22, Leitz, Wetzlar, Germany). In order to have a quantitative estimation of damage caused by ischemia/reperfusion, sections (n = 6 for each animal) were scored by two independent observers blind to the experimental protocol. The following morphological criteria were used for scoring: 0, no damage; 1, (mild) focal epithelial oedema and necrosis; 2, (moderate) diffuse swelling and necrosis of the villi; 3, (severe) necrosis with evidence of neutrophil infiltration in the submucosa; and 4, (major) widespread necrosis with massive neutrophil infiltration and evidence of hemorrhage.
Determination of myeloperoxidase (MPO) activity
MPO activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described (18). After weighing, each piece of intestine tissues was homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 μM of peroxide per minute at 37°C and was expressed in milliunits per gram weight of wet tissue.
Determination of malondialdehyde (MDA) levels
The levels of MDA in the ileum tissue were determined as an indicator of lipid peroxidation (19). Ileum tissue, collected at the specified time, was homogenized in 1.15% KCl solution. An aliquot (100 μL) of the homogenate was added to a reaction mixture containing 200 μL of 8.1% SDS, 1500 μL of 20% acetic acid (pH 3.5), 1500 μL of 0.8% thiobarbituric acid, and 700 μL distilled water. Samples were then boiled for 1 h at 95°C and were centrifuged at 3000 g for 10 min. The absorbance of the supernatant was measured by spectrophotometry at 650 nm.
Measurement of plasma levels of cytokines
TNF-α, IL-6, and IL-1β levels were evaluated in plasma samples. The assay was carried out by using a colorimetric commercial kit (Calbiochem-Novabiochem Corporation, Milan, Italy). The ELISA has a lower detection level of 10 pg/mL.
Measurement of IκB-α
A 7- to 8-cm section of ileum was excised and the mucosal surface was exposed, rinsed with sterile saline, and the epithelium was scraped with a No. 10 scalpel blade. The resultant cell/tissue pellet was prepared as described (20). Briefly, harvested cells (2 × 107) were washed twice with ice-cold PBS and centrifuged at 180 g for 10 min at 4°C. The cell pellet was resuspended in 100 μL of ice-cold hypotonic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride, 1.5 μg/mL soybean trypsin inhibitor, 7 μg/mL pepstatin A, 5 μg/mL leupeptin, 0.1 mM benzamidine, and 0.5 mM dithiothreitol) and was incubated in ice for 15 min. The cells were lysed by rapid passage through a syringe needle five or six times, and the cytoplasmic fraction was then obtained by centrifugation for 1 min at 13,000 g for 1 min. Protein concentration was determined with a protein assay kit (Bio-Rad, Hercules, CA). Immunoblotting analysis of IκB-α proteins was performed on cytosolic fraction. Cytosolic fraction proteins were mixed with gel loading buffer (50 mM Tris/10% SDS/10% glycerol/10% 2-mercaptoethanol/2 mg bromphenol per milliliter) in a ratio of 1:1, boiled for 3 min, and centrifuged at 10,000 g for 10 min. Protein concentration was determined and equivalent amounts (75 μg) of each sample were electrophoresed in a 12% discontinuous polyacrylamide minigel. The proteins were transferred onto nitrocellulose membranes according to the manufacturer's instructions (Bio-Rad). The membranes were saturated by incubation at 4°C overnight with 10% non-fat dry milk in PBS and were then incubated with anti-IκB-α (1:1000) for 1 h at room temperature. The membranes were washed three times with 1% (w/v) Triton X-100 in PBS and then were incubated with anti-rabbit immunoglobulins coupled to peroxidase (1:1000). The immune complexes were visualized by the ECL chemiluminescence method (Amersham, Piscataway, NJ).
Reagents
GW274150 was obtained from Alexis (Milan, Italy). Biotin blocking kit, biotin-conjugated goat anti-rabbit IgG, and avidin-biotin peroxidase complex were obtained from Vector Laboratories (Burlingame, CA). Primary anti-nitrotyrosine antibody was purchased from Upstate Biotech (Saranac Lake, NY). Primary monoclonal P-Selectin (CD62P) or ICAM-1 (CD54) for immunohistochemistry were purchased from BD PharMingen (San Diego, CA). Reagents and secondary and nonspecific IgG antibody for immunohistochemical analysis were also from Vector Laboratories. Primary monoclonal anti-poly(ADP-ribose) antibody was purchased from Alexis. All other reagents and compounds used were obtained from Sigma Chemical Company (St. Louis, MO).
Statistical analysis
All values in the figures and text are expressed as means ± SEM of n observations, where n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. Data sets were examined by one- and two-way analysis of variance, and individual group means were then compared with Student's unpaired t test. Non-parametric data were analyzed with the Fisher's exact test. A P value less than 0.05 was considered significant.
RESULTS
Role of iNOS on mortality after splanchnic ischemia/reperfusion
To study the clinical situation of mesenteric infarction, mice were subjected to 45 min of occlusion followed by reperfusion of the superior mesenteric artery and celiac trunk. In two preliminary studies, iNOSWT mice treated with GW274150 and iNOS-KO mice did not exhibit any difference in the rate of mortality in comparison with wild-type control mice, and all died within 45 to 60 min after the onset of reperfusion (Fig. 1A). In a subsequent study, we evaluated the survival in a less severe model of intestinal injury obtained by selectively occluding the superior mesenteric artery only for 45 min. Using this model, we found that the mortality of iNOSWT mice treated with GW274150 and iNOS-KO mice was substantially lower than in the respective wild-type controls (Fig. 1B). Based on these findings, all further experiments were performed in mice subjected to 45 min occlusion of the superior mesenteric artery only followed by reperfusion for 4 h.
;)
Fig. 1: Survival was monitored for 4 h after severe ischemia/reperfusion injury was induced by clamping, for 45 min, the superior mesenteric artery and the celiac trunk (A). Survival was also monitored for 4 h after a milder ischemia/reperfusion injury was induced by occluding, for 45 min, the superior mesenteric artery only (B). Data are means ± SEM of 10 mice for each group. * P < 0.01 represents significant reduction of the various parameters in the group in which iNOS activity was inhibited or iNOS was absent.
Splanchnic ischemia/reperfusion injury is reduced in iNOS-KO mice
In sham wild-type and iNOS-KO mice, the histological structure of the gastrointestinal tract were typical of a normal architecture (Fig. 2a). In wild-type mice, splanchnic ischemia/reperfusion resulted in tissue injury mainly localized to the small intestine. Further histological examination of the tissue demonstrated damage localized to the villi and was associated with infiltration of the inflammatory cells in the mucosa as well as tissue hemorrhage (see Fig. 2b for representative section). The degree of the tissue injury (on an arbitrary score ranging from 0 to 4) was 3.24 ± 0.09. The damage score for iNOS-KO mice was significantly lower (1.63 ± 0.08) than that obtained from wild-type mice (P < 0.001) as well as the histological observation (Fig. 2c). Treatment of wild-type mice with the iNOS inhibitor GW274150 also resulted in a significant reduction of the damage score (1.91 ± 0.01), as well as a reduction in the histological signs of tissue injury (Fig. 2d).
Fig. 2: Distal ileum section from a sham iNOSWT mouse (a) demonstrating the normal architecture of the intestinal epithelium and wall. Distal ileum section from a SMO iNOSWT mouse showed inflammatory cells infiltration extending through the wall and concentrated below the epithelial layer and necrosis of the epithelium at the villus tips (b). Distal ileum from a SMO iNOSKO mouse shows reduced SMO-induced ileum injury (c). GW274150-treatment of SMO iNOSWT mice significantly prevented ileum damage (d). Original magnification: ×125. Figure is representative of at least three experiments performed on different experimental days.
iNOS activity is required for the upregulation of cytokines levels during splanchnic ischemia/reperfusion
Splanchnic ischemia/reperfusion results in the upregulation of proinflammatory cascades in the intestine as well as in other organs (21). The inflammatory response includes the expression of iNOS in the late phase of reperfusion (22). In this study, we found that the activity of iNOS in the intestine increased by 5- to 6-fold over sham-treated animals (P = 0.001) after 4 h of reperfusion (Fig. 3). To determine whether an enhanced formation of NO by iNOS participates in the upregulation of cytokine expression after splanchnic ischemia/reperfusion, wild-type mice treated with GW274150 and iNOS-KO mice were subjected to splanchnic ischemia/reperfusion. IL-6, TNF-α, and IL-β levels have been previously shown to be consistently elevated in the plasma from splanchnic ischemia/reperfusion (21) and, in the case of IL-6, may contribute to leukocyte recruitment and activation (22). IL-6, TNF-α, and IL-β plasma levels were significantly increased in comparison with sham animals (Fig. 4). This upregulation was significantly reduced in the iNOS-KO mice. Therefore, the administration of GW274150 to wild-type mice significantly reduced the increase of the cytokines levels (Fig. 4).
Fig. 3: iNOS activity at 4 h after SMO. iNOS activity in SMO-treated iNOSWT mice was significantly increased vs. sham group. No significant iNOS activity was found in SMO iNOSKO mice. GW274150 treatment of SMO-shocked iNOSWT mice show a significant reduction iNOS activity. Data are means ± SEM of 10 mice for each group. * P < 0.01 represents significant reduction of the various parameters in the group in which iNOS was inhibited or absent.
Fig. 4: TNF-α (A), IL1-β (B), and IL-6 (C) plasma levels. iNOSWT mice show a significant production of cytokines after 4 h reperfusion. Cytokine levels were significantly reduced in iNOSKO mice and in the iNOSWT mice treated with GW274150. Data are means ± SEM of 10 mice for each group. * P < 0.01 represents significant reduction of the various parameters in the group in which iNOS was inhibited or absent.
P-selectin/ICAM-1 expression and neutrophil infiltration are reduced in iNOS-KO mice
Assessment of neutrophil infiltration into the ileum was performed by measuring the activity of MPO, an enzyme that is contained in and is specific for PMN lysosomes. MPO activity was significantly elevated after splanchnic ischemia/reperfusion in wild-type mice (Fig. 5A). The elevation of the MPO activity was associated with the increase of immunohistochemical staining for P-selectin (Fig. 6b) and ICAM-1 (Fig. 7b) in the injured splanchnic tissue. In wild-type mice treated with GW274150 and iNOS-KO mice, tissue MPO activity (Fig. 5A) was markedly reduced in comparison with those of wild-type control animals. No positive staining for P-selectin was found in the intestine of iNOS-KO mice (Fig. 6c) and in wild-type mice treated with GW274150 subjected to splanchnic ischemia/reperfusion (Fig. 6d). Similarly, less positive for ICAM-1 was observed in the intestine of iNOS-KO mice (Fig. 7c) and in wild-type mice treated with GW274150 subjected to splanchnic ischemia/reperfusion (Fig. 7d). Please note that staining of intestine tissue sections obtained from sham-operated mice with anti-ICAM-1 antibody showed a specific staining along vessels, demonstrating that ICAM-1 is constitutively expressed (Fig. 7a). No positive staining for P-selectin was found in intestine tissue section from sham-operated mice (Fig. 6c).
Fig. 5: MPO activity (A) and MDA levels (B) in the reperfused intestine of SMO mice sacrificed after 4 h reperfusion. MPO activity and MDA levels were significantly increased in SMO iNOSWT mice in comparison with sham. iNOSKO mice and the iNOSWT mice treated with GW274150 show a significant reduction of MPO activity and MDA levels. Data are means ± SEM of 10 mice for each group. * P < 0.01 represents significant reduction of the various parameters in the group in which iNOS was inhibited or absent.
Fig. 6: Ileum section from sham-operated mice (a) revealed no P-selectin staining. Section obtained from SMO iNOSWT mice showed intense positive staining for P-selectin (b). The degree of positive staining was markedly reduced in tissue section obtained from iNOSKO mice (c) and the iNOSWT mice treated with GW274150 (d). Original magnification: ×150. Figure is representative of at least three experiments performed on different experimental days.
Fig. 7: Control tissue from sham-operated mice (a) showed a dark brown staining of endothelium of blood vessels, indicating the presence of constitutive ICAM-1 protein. Ischemia and reperfusion in iNOSWT mice induced an increase of the positive staining for ICAM-1 along the endothelium wall (b). In iNOSKO mice (c) and in iNOSWT mice treated with GW274150 (d) subjected to SMO, there was no increase of immunostaining for ICAM-1, which was present only along the endothelium wall. Original magnification: ×150. This figure is representative of at least three experiments performed on different experimental days.
Absence of iNOS gene reduced lipid peroxidation, nitrotyrosine, and PAR formation
The release of free radicals and oxidant molecules during the early period of reperfusion has been suggested to contribute significantly to the tissue necrosis and mucosal dysfunction (2–4). Splanchnic ischemia/reperfusion injury of wild-type mice was characterized by an increase in tissue MDA, indicative of lipid peroxidation (Fig. 5B). Furthermore, positive staining for nitrotyrosine, a marker of nitrosative injury, was found on epithelial and infiltrated inflammatory cells in the injured small intestine of wild-type mice subjected to SMO shock (Fig. 8b). In wild-type mice treated with GW274150 and iNOS-KO mice, tissue MDA levels (Fig. 5B) were markedly reduced in comparison with those of wild-type control animals. No positive nitrotyrosine staining was found in the intestine of iNOS-KO mice (Fig. 8c) and in wild-type mice treated with GW274150 subjected to splanchnic ischemia/reperfusion (Fig. 8d).
Fig. 8: Ileum section from sham-operated mice (a) revealed no nitrotyrosine staining. Section obtained from SMO iNOSWT mice showed intense positive staining for nitrotyrosine (b). The degree of positive staining was markedly reduced in tissue section obtained from iNOSKO mice (c) and the iNOSWT mice treated with GW274150 (d). Original magnification: ×150. This figure is representative of at least three experiments performed on different experimental days.
Intestinal sections were also taken in order to determine the immunohistological staining for PAR proteins (an indicator of PAR protein activation). Immunohistochemical analysis of intestinal sections obtained from wild-type mice subjected to splanchnic ischemia/reperfusion revealed a positive staining for PAR, which was primarily localized in inflammatory cells (Fig. 9b). In contrast, significantly less positive PAR staining was found in the intestine of iNOS-KO mice (Fig. 9c) and in wild-type mice treated with GW274150 also subjected to splanchnic ischemia/reperfusion (Fig. 9d). Please note that there was no staining for either nitrotyrosine or PAR in intestine obtained from sham-operated mice (Figs. 8a and 9a).
Fig. 9: Ileum section from sham-operated mice (a) revealed no PAR staining. Section obtained from SMO iNOSWT mice showed intense positive staining for PAR (b). The degree of positive staining was markedly reduced in tissue section obtained from iNOSKO mice (c) and the iNOSWT mice treated with GW274150 (d). Original magnification: ×150. This figure is representative of at least three experiments performed on different experimental days.
Degradation of IκB-α
The level of IκB-α protein in the cytosolic fractions of intestinal epithelial cells was investigated using Western blot analysis. A basal level of IκB-α was detectable in the cytosolic fraction of cell/tissue pellet from sham-operated mice, whereas 4 h after reperfusion, IκB-α disappeared in the cell/tissue pellet collected from wild-type mice subjected to splanchnic ischemia/reperfusion. A significant prevention of IκB-α degradation was observed in the cell/tissue pellet collected from iNOS-KO mice subjected to splanchnic ischemia/reperfusion as well as in wild-type mice treated with GW274150 and also subjected to splanchnic ischemia/reperfusion. In fact, the IκB-α band remained unchanged after 4 h of reperfusion (Fig. 10).
Fig. 10: Western blot analysis shows the effect of iNOS inhibition or absence on degradation of IkB-α in epithelial cells collected after 4 h reperfusion. Control, Basal level of IκB-α band was present in the cytosolic fraction of unstimulated cells. SMO/R, IκB-α band has disappeared from the cytosolic fraction of cells collected from SMO iNOSWT mice. SMO/R iNOSKO, IκB-α band remained unchanged in the cytosolic fraction of cells collected from SMO iNOSKO mice. SMO/R + GW274150, IκB-α band remained unchanged in the cytosolic fraction of cells collected from SMO iNOSWT mice, which received GW274150. The data illustrated are representative of a total of three separate experiments.
DISCUSSION
We report here that mice with a targeted deletion of the iNOS gene (iNOS-KO) or wild-type mice treated with the novel, potent, and selective iNOS inhibitor GW274150, are protected against the pathological changes caused by ischemia/reperfusion injury of the gut. Thus, we propose that an enhanced formation of NO by iNOS contributes to the pathophysiology of ischemia/reperfusion injury of the gut.
It has been proposed that the L-arginine/NO pathway plays an important role in the pathogenesis of ischemia/reperfusion injury and shock. In the latter, the production of large amount of NO by iNOS contributes to the delayed vascular decompensation and to the hyporeactivity of the vasculature to vasoconstrictor agents observed in several experimental models of circulatory shock (5). However, the role of iNOS in intestinal ischemia/reperfusion injury is still controversial. Previous findings have indicated that an enhanced formation of NO by iNOS might play an important role in the pathophysiology of splanchnic ischemia/reperfusion (25). Indeed, this hypothesis is also supported by the finding that 1400-W, a selective inhibitor of iNOS (26), was able to protect against the pathological sequel associated with splanchnic artery occlusion (27). In contrast, the results of a previous study have suggested that it is unlikely that iNOS play a key role in this model of splanchnic ischemia/reperfusion. It should be noted that in that study (28), the animals were sacrificed at a relatively short time after reperfusion (1 h), which is a relatively short time to allow for the expression of iNOS.
Splanchnic ischemia/reperfusion injury is characterized by an intense inflammatory infiltrate found predominantly in the mucosa and submucosa, causing epithelial destruction via the release of ROS, RNS, and cytokines (2–4). Endothelial adhesion molecules are important regulators of neutrophil adhesion, activation, and trans-endothelial migration. During the early phase of reperfusion, P-selectin is rapidly released to the endothelial surface from preformed storage pools after exposure to certain stimuli such as H2O2, thrombin, histamine, or complement, and it allows the leukocytes to roll along the endothelium (29). ICAM-1, constitutively expressed on the surface of endothelial cells, is subsequently involved in the adhesion of neutrophils (27).
Intestinal ischemia followed by reperfusion for 4 h resulted in the upregulation of P-selectin on the endothelium of small vessels mainly in the lamina propria, as well as the upregulation of ICAM-1 on endothelial and epithelial cells. However, we demonstrated a significant reduction in the expression of P-selectin and ICAM-1 in iNOS-KO mice in comparison with wild-type mice after 4 h of reperfusion. Interestingly, we found that constitutive expression of ICAM-1 did not differ between sham iNOS-KO and wild-type mice in the vasculature of the small intestine. Taken together with the finding of a marked reduction of the neutrophil infiltration in the intestine of iNOS-KO mice, these data suggest that iNOS modulates adhesion molecule expression and subsequent neutrophil infiltration. In addition, NO appears to play an inhibitory role in the expression of adhesion molecules during ischemia/reperfusion injury, regulating neutrophil recruitment both at the rolling and firm adhesion phase.
The reduction of leukocyte infiltration in iNOS-KO mice and in wild-type mice treated with GW274150 correlated well with the reduction of tissue damage after 4 h of reperfusion, as evaluated by histological examination as well as the intestinal lipid peroxidation assessed by thiobarbituric acid-reactant MDA. A significant reduction of the immunostaining of nitrotyrosine formation also suggested that a structural alteration of mucosal proteins had occurred, most probably due to the formation of RNS and that iNOS may also play a role in this regard. In fact, less nitrotyrosine staining was found in iNOS-KO and in wild-type mice treated with GW274150. Recent evidence indicates that several chemical reactions, involving nitrite, peroxynitrite, hypochlorous acid, and peroxidases can induce tyrosine nitration and may contribute to tissue damage (30).
Therefore, using either the iNOS inhibitor GW274150 or iNOS-KO mice, we have demonstrated that iNOS contributes to the induced expression of the cytokines during splanchnic ischemia/reperfusion. The iNOS-dependent increase in cytokines levels is associated with an iNOS-dependent increase in NF-κB activation in the intestinal tissues. These data provide compelling evidence that iNOS is in part responsible for the activation of inflammatory cascades as well as intestine damage during splanchnic ischemia/reperfusion.
The mechanism of the upregulation of iNOS during splanchnic ischemia/reperfusion is unclear, but could involve hypoxia (5) or the action of cytokines. We have shown that iNOS activity increases in the intestine, whereas others have suggested that iNOS contributes to the initial vascular decompensation in hemorrhagic shock (5) and have suggested that the iNOS expression pattern is consistent with the possibility that iNOS contributes to the progressive vascular dysfunction seen with sustained shock (31). The data in this study support the notion that NO can increase cytokine expression via the activation of NF-κB. Recently, using iNOS-KO mice, it has been demonstrated that NO from iNOS may regulate cytokine formation and NF-κB activation in hemorrhagic shock tissues (32). Thus, it is likely that the NO-mediated signalling events that are initiated in early phases of reperfusion result in the rapid activation of downstream cascades. Although NO produced by the constitutive NO synthase has well-documented signalling functions in many systems, our novel observations provide strong evidence that induced NO also participates in cell signalling events in splanchnic ischemia/reperfusion. That iNOS regulation of inflammatory gene expression is perhaps a more generalized phenomenon is supported by a recent observation that the upregulation of interferon and the response to IL-12 after Leishmania major infection is dependent on iNOS (33). However, our results do not exclude the possibility that the observed differences in both the GW274150-treated wild-type mice and the iNOS-KO mice are due to other mechanisms such us changes in organ perfusion and oxygen delivery resulting from reduced NO availability. NO is known to act as a signalling molecule in other circumstances either by activation of soluble guanylyl cyclase resulting in elevated cyclic guanosine 3´,5´ monophosphate (cGMP) (10) or via S-nitrosylation of proteins containing cysteine residues (34). Significant differences in cytokine mRNA levels (35) and transcriptional factor activation (36) between the sham and ischemia/reperfusion groups was observed only after reperfusion, indicating that reperfusion was required. This suggests that redox-sensitive mechanisms were responsible for the NO-mediated signalling. Lander et al. (5) have shown that NO activates p21ras via S-nitrosylation and that this occurs more efficiently in human T cells subjected to oxidative stress. Downstream events include p38 kinase activation and NF-κB activation. In splanchnic ischemia/reperfusion, tissues are subjected to redox stress by hypoxia and oxygen radical production, making S-nitrosylation of p21ras a reasonable candidate mechanism. One report demonstrates a role for increased phosphatidic acid in the signalling cascade involved in macrophage cytokine synthesis after hemorrhagic shock in mice (36). A relationship between phosphatidic acid and NO is not apparent at this time. We have previously shown that non-specific NO synthase inhibition increases organ injury in intestine ischemia/reperfusion injury (27), whereas here we demonstrate that the suppression of inflammation associated with selective iNOS inhibition results in a decrease in intestine injury. Taken together, the findings suggest that induction of iNOS and subsequent production of NO contribute to splanchnic ischemia/reperfusion. The proinflammatory properties of iNOS include the modulation of secretion of proinflammatory cytokines, endothelial expression of P-selectin and ICAM-1, with consequent stimulation of neutrophil infiltration and related oxidative and nitrosative stress. This proinflammatory effect may be also attributed to a modulation of the cellular signalling mechanisms mediated by NF-κB. These novel pathophysiological insights may provide a new basis for the development of therapies for limiting ischemia/reperfusion injury.
ACKNOWLEDGMENTS
The authors would like to thank Fabio Giuffrè and Carmelo La Spada for their excellent technical assistance during this study, Mrs. Caterina Cutrona for secretarial assistance, and Miss Valentina Malvagni for editorial assistance with the manuscript. Christoph Thiemermann is a Senior Fellow of the British Heart Foundation (FS 96/018).
REFERENCES
1. Cappel MS: Intestinal (mesenteric) vasculopathy. I. Acute superior mesenteric arteriopathy and venopathy. Gastroenterol Clin North Am 27:783–790, 1998.
2. Squadrito F, Altavilla D, Canale P, Ioculano M, Campo GM, Ammendolia L, Ferlito M, Zingarelli B, Squadrito G, Saitta A, Caputi AP: Participation of tumour necrosis factor and nitric oxide in the mediation of vascular dysfunction in splanchnic artery occlusion shock. Br J Pharmacol 113:1153–1158, 1994.
3. Cuzzocrea S, Zingarelli B, Costantino G, Szabò A, Salzman AL, Caputi AP, Szabò C: Beneficial effects of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase in a rat model of splanchnic artery occlusion and reperfusion. Br J Pharmacol 121:1065–1074, 1997.
4. Lander HM: An essential role for
free radicals and derived species in signal transduction. FASEB J 11:118–124, 1997.
5. Moncada S, Higgs EA: The L-arginine-nitric oxide pathway. N Engl J Med 329:2002–2012, 1993.
6. Nathan C: Nitric oxide as a secretory product of mammalian cells. FASEB J 6:3051–3064, 1992.
7. Kubes P: Ischemia-reperfusion in feline small intestine: a role for nitric oxide. Am J Physiol 264:G143–G149, 1993.
8. Aoki N, Johnson III, G Lefer AM: Beneficial effects of two forms of NO administration in feline splanchnic artery occlusion shock. Am J Physiol 258:G275–G281, 1990.
9. Payane D, Kubes P: Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol 265:G189–G195, 1993.
10. Carey C, Siegfried MR, Ma XL, Weyrich AS, Lefer AM: Antishock and endothelial protective actions of a NO donor in mesenteric ischemia and reperfusion. Circ Shock 38:209–216, 1992.
11. Szabó C: The pathophysiological role of
peroxynitrite in shock, inflammation and ischemia-reperfusion injury. Shock 6:79–88, 1996.
12. Szabó C, Thiemermann C, Wu CC, Perretti M, Vane JR: Inhibition of nitric oxide synthase induction by endogenous glucocorticoids accounts for endotoxin tolerance in vivo. Proc Natl Acad Sci USA 91:271–275, 1994.
13. Alderton W, Angell A, Clayton N, Craig C, Dawson J, Frend A, McGill J, Mangel A, Moncada S, Rees D, Russell L, Schwartz S, Waslidge N, Knowles R: GW274150 is a potent, long-acting, highly selective inhibitor of
iNOS (NOS-2) with therapeutic potential in post-operative ileus.
Acta Physiol Scand 167(Abstract):36, 1999.
14. Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 357:593–615, 2001.
15. Alderton W, Angell A, Clayton N, Craig C, Dawson J, Frend A, McGill J, Mangel A, Moncada S, Rees D, et al.: GW274150 is a potent, long-acting, highly selective inhibitor of inducible nitric oxide synthase (NOS-2 with therapeutic potential in post-operative ileus. In Moncada S, Gustaffson LE, Wiklund NP, Higgs EA (eds):
Biology of Nitric Oxide, Vol 7. London: Portland Press, 2000, 22 pp.
16. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, Hutchinson N: Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641–650, 1995.
17. Cuzzocrea S, Caputi AP, Zingarelli B:
Peroxynitrite-mediated DNA strand breakage activates poly (ADP-ribose) synthetase and causes cellular energy depletion in carrageenan-induced pleurisy. Immunology 93:96–101, 1998.
18. Mullane KM, Westlin W, Kraemer R: Activated neutrophils release mediators that may contribute to myocardial injury and dysfunction associated with ischemia and reperfusion. Ann NY Acad Sci 524:103–121, 1988.
19. Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358, 1979.
20. Schreiber E, Metthias P, Muller MM, Shaffner W: Rapid detection of octamer binding proteins with “mini-extracts” prepared from a small number of cells. Nucleic Acids Res 17:6420, 1989.
21. Lefer AM, Tsao PS, Ma XL: Shock- and ischemia-induced mechanisms of impairment of endothelium-mediated vasodilation. Chest 100:1605–1635, 1991.
22. Suzuki Y, Deitch EA, Mishima S, Lu Q, Xu D: Inducible nitric oxide synthase gene knockout mice have increased resistance to gut injury and bacterial translocation after an intestinal ischemia-reperfusion injury. Crit Care Med 28:3692–3696, 2000.
Cuzzocrea S, Misko TP, Costantino G, Mazzon E, Micali A, Caputi AP, Macarthur H, Salvemini D: Beneficial effects of
peroxynitrite decomposition catalyst in a rat model of splanchnic artery occlusion and reperfusion. FASEB J 14:1061–1072, 2000.
Romano M, Sironi M, Toniatti C, Polentarutti N, Fruscella P, Ghezzi P, Faggioni R, Luini W, van Hinsberg V, Sozzani S, Bussolino F, Poli V, Ciliberto G, Mantovani A: Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 6:315–325, 1997.
25. Squadrito F, Altavilla D, Squadrito G, Campo GM, Ioculano M, Canale P, Rossi F, Saitta A, Caputi AP: Effects of
S-ethylisothiurea, a potent inhibitor of nitric oxide synthase, alone or in combination with a nitric oxide donor in a splanchnic artery occlusion shock. Br J Pharmacol 119:23–28, 1996.
26. Garvey EP, Oplinger JA, Furfine ES, Kiff RJ, Laszlo F, Whittle BJ, Knowles RG: 1400W is a slow, tight binding and high selective inhibitor of inducible nitric oxide synthase in vitro and in vivo. J Biol Chem 21:4959–4963, 1997.
27. Squadrito F, Altavilla D, Squadrito G, Ferlito M, Deodato B, Arlotta M, Minutoli L, Campo GM, Bova A, Quartarone C, Urna G, Sardella A, Saitta A, Caputi AP: Protective effects of cyclosporin-A in splanchnic artery occlusion shock. Br J Pharmacol 130:339–344, 2000.
28. Cuzzocrea S, Zingarelli B, Caputi AP: Role of constitutive nitric oxide synthase and
peroxynitrite production in a rat model of splanchnic artery occlusion shock. Life Sci 63:789–799, 1998.
29. Lawrence MB, Springer TA: Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65:859–873, 1991.
30. Eiserich JP, Cross CE, Jones AD, Halliwell B, van der Vliet A: Formation of nitrating and chlorinating species by reaction of nitrite with hypochlorous acid: a novel mechanism for nitric oxide-mediated protein modification. J Biol Chem 271:19199–19208, 1996.
31. Kelly E, Shah NS, Morgan NN, Watkins SC, Peitzman AB, Billiar TR: Physiologic and molecular characterization of the role of nitric oxide in hemorrhagic shock: evidence that type II nitric oxide synthase does not regulate vascular decompensation. Shock 7:157–163, 1997.
32. Hierholzer C, Harbrecht B, Menezes JM, Kane J, MacMicking J, Nathan CF, Peitzman AB, Billiar TR: Tweardy Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock. J Exp Med 187:917–928, 1998.
33. Diefenbach A, Schindler H, Donhauser N, Lorenz E, Laskay T, MacMiking J, Rollinghoff M, Gresser I, Bogdan C: Type 1 interferon (IFN-α/β) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite. Immunity 8:77–87, 1998.
34. Hierholzer C, Kelly E, Billiar TR, Tweardy DJ: Granulocyte colony-stimulating factor (G-CSF) in hemorrhagic shock requires both the ischemic and resuscitation phase. Arch Orth Trauma Surg 116:173–176, 1997.
35. Hierholzer C, Kalff J, Kim Y, Billiar TR, Tweardy DJ: G-CSF and IL-6 are produced in the lung in hemorrhagic shock where they contribute to the recruitment and activation of neutrophils. In Faist E (ed):
4th International Congress on the Immune Consequences of Trauma, Shock, and Sepsis: Mechanism and Therapeutic Approaches. Bologna: Monduzzi, 1997.
36. Hierholzer C, Kalff J, Billiard T, Tweardy D: Activation of STT proteins in the lung of rats following resuscitation from hemorrhagic shock.
Arch Orth Trauma Surg (in press).
Lander HM, Jacovina AT, Davis RJ, Tauras JM: Differential activation of mitogen-activated protein kinases by nitric oxide-related species. J Biol Chem 271:19705–19709, 1996.
