Chimeric SOD2/3 inhibits at the endothelial-neutrophil interface to limit vascular dysfunction in ischemia-reperfusion
Claudine S. Bonder,1
Derrice Knight,1
Daniel Hernandez-Saavedra,2
Joe M. McCord,2 and
Paul Kubes1
1Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1; and 2Webb-Waring Institute For Biomedical Research, University of Colorado Health Sciences Center, Denver, Colorado 80262
Submitted 29 January 2004
; accepted in final form 7 April 2004
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ABSTRACT
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After an ischemic episode, reperfusion causes profound oxidative stress in the vasculature of the afflicted tissue/organ. The dysregulated accumulation of reactive oxygen species (ROS), such as superoxide, has been closely linked to the production and release of proinflammatory mediators, a profound increase in adhesion molecule expression by the vascular endothelium, and infiltration of neutrophils during ischemia-reperfusion (I/R). Superoxide dismutase (SOD) has been shown to protect tissues and organs against I/R-induced injury; however, the drug had to be continuously perfused or kidneys had to be occluded to prevent clearance. We used intravital microscopy, a system that allowed us to visualize neutrophil-endothelial interactions within the mesenteric postcapillary venules of cats subjected to I/R and tested the hypothesis that I/R-induced neutrophil recruitment was inhibited by treatment with SOD2/3. SOD2/3 is a chimeric fusion gene product that contains the mature SOD2 as well as the COOH-terminal "tail" of SOD3 and, unlike the three naturally occurring SODs (SOD1, SOD2, and SOD3), which bear a net negative charge at pH 7.4, SOD2/3 is positively charged and physiologically stable. Our results suggest that not only does SOD2/3 have a much greater efficacy in vivo than the native human SOD2, but its administration prevents I/R-induced neutrophil-endothelial cell interactions and microvascular dysfunction. Moreover, our data support the hypothesis that reactive oxidants mediate I/R-induced injury and that the chimeric recombinant SOD2/3 has the potential to be a therapeutic agent against this debilitating illness.
superoxide dismutase; in vivo; endothelium
ALTHOUGH OXYGEN IS INDISPENSABLE for normal cellular function, it can be transformed into highly reactive forms, i.e., reactive oxygen species (ROS), which are often toxic to the cell. It is well recognized that the formation of ROS, after an ischemic episode, is directly associated with ischemia-reperfusion (I/R)-induced injury. With reperfusion, the production of ROS, such as superoxide (O2), is rapid and has been shown to come directly from the vascular endothelium via enzymes such as xanthine oxidase. The excessive production of O2 during I/R causes profound oxidative stress on the endothelium, which then causes the production and release of proinflammatory mediators. The oxidative stress also causes an increase in expression of adhesion molecules on the endothelial cell surface. With these responses combined, there is profound recruitment of neutrophils from the circulation to the afflicted tissue. The direct role for neutrophils in I/R-induced injury has been demonstrated by many investigators. For example, neutrophils have been shown to infiltrate postischemic tissue (10, 26, 44), depletion of neutrophils from the circulation significantly impairs reperfusion-induced tissue injury (15, 44), and reagents that interfere with neutrophil infiltration into postischemic vessels provide protection against reperfusion injury (12, 20, 27, 51).
One enzyme that is part of the cell's arsenal against ROS is superoxide dismutase (SOD) (28, 31, 34). In humans, three isozymes of SOD have been extensively characterized: the cytosolic CuZnSOD, also known as SOD1 (34); the mitochondrial MnSOD or SOD2 (33); and an extracellular SOD or SOD3 (28). SOD3 is found extracellularly in relatively few tissues and exists overall at a much lower concentration than either intracellular enzyme (29, 30, 42). Under normal circumstances, intracellularly generated O2 is efficiently handled by the cytosolic SOD1 and mitochondrial SOD2. SOD3, which binds to endothelial cell surfaces via its hydrophilic positively charged tail (45), protects endothelial cell surfaces from extracellular O2 attack. Under pathological conditions, when large amounts of reactive oxidants are produced, the overproduction of endogenous O2 overwhelms the SOD levels. In addition, SOD3 is cleaved from the endothelial cell surface by proteases, which renders the endothelium susceptible to O2 attack (35). Given the affinity of SOD3 for the endothelium, there is a clear advantage of utilizing SOD3 to combat O2 attack during I/R. Unfortunately, high-level expression of recombinant SOD3 has not been successful (14, 48).
To provide a therapeutic equivalent of SOD3, a fusion gene product was generated that consists of the mature human SOD2 and the COOH-terminal 26-amino acid "tail" from SOD3. The resulting recombinant SOD2/3 protein can be purified readily and in high yield from Escherichia coli, retains the enzymatic activity of SOD2, and binds heparin with high affinity (9). Recent applications of SOD2/3 have shown its effectiveness in inflammation. For example, the chimeric SOD2/3 provided almost complete protection from neutrophil migration and lung leakage caused by IL-1 (9). SOD2/3 has also been effective in reducing inflammation in a carrageenan-induced foot edema model (9). Similarly, it has inhibited endotoxin-induced platelet-endothelial cell adhesion in intestinal venules (5). The aim of this study was to investigate the effect of SOD2/3 on leukocyte-endothelial cell interactions that take place during I/R. Using intravital microscopy, we investigated leukocyte recruitment to the mesenteric postcapillary venules, inasmuch as it allows real-time quantitative analysis of cell kinetics (rolling flux, rolling velocity, adhesion and emigration). Previous work has shown that SOD is rapidly cleared by the kidneys. Our results suggest that SOD2/3 was very effective in vivo and prevented leukocyte-endothelial cell interactions and microvascular dysfunction.
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MATERIALS AND METHODS
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Animals and reagents.
All cats were maintained in the animal facility at the University of Calgary until they weighed 1.22.4 kg, at which point they were used. Animal protocols were approved by the University of Calgary Animal Care Committee and met the Canadian Guidelines for Animal Research. Some cats received the chimeric recombinant form of SOD, SOD2/3, with a specific activity of 3,000 U/mg (9, 38). Native human recombinant SOD2, with a comparable activity of 3,500 U/mg, was used as a control. SOD2 or SOD2/3 was administered via the left jugular vein at 0.5 mg/animal in 1 ml of sterile saline 5 min before reperfusion.
Immunohistochemistry.
Small (1 cm long) sections of ileum were excised before induction of ischemia as well as 1 and 4 h after reperfusion and fixed in 10% formalin (Sigma). Formalin-fixed tissues were embedded in paraffin. Sections (4 µm thick) were cut and stained using hematoxylin and eosin in a standard fashion. Leukocyte infiltration and villus length were visualized under a magnification of x40.
Intravital microscopic studies.
The surgical preparation used in this study is the same as that described previously (7, 19). Briefly, age-matched cats (1.22.4 kg) were fasted for 12 h and initially anesthetized with ketamine hydrochloride (75 mg im). The jugular vein was cannulated, and anesthesia was maintained by administration of pentobarbital sodium. A tracheotomy was performed to support breathing by artificial ventilation. Systemic arterial pressure was monitored by a pressure transducer (Statham P23A, Gould, Oxnard, CA) connected to a catheter in the left carotid artery. A midline abdominal incision was made, and a segment of small intestine was isolated from the ligament of Treitz to the ileocecal valve. The remainder of the small and large intestines was extirpated. Body temperature was maintained at 37°C using an infrared heat lamp. All exposed tissues were moistened with saline-soaked gauze to prevent evaporation. Heparin sodium (10,000 U; Elkins-Sinn, Cherry Hill, NJ) was administered, and an arterial circuit was established between the superior mesenteric artery (SMA) and the left femoral artery. SMA blood flow was continuously monitored using an electromagnetic flowmeter (Carolina Medical Electronics, King, NC). Blood pressures were continuously recorded with a physiological recorder (Grass Instruments, Quincy, MA).
Cats were placed in a supine position on an adjustable Plexiglas microscope stage, and a segment of midjejunum was exteriorized through the abdominal incision. The mesentery was prepared for in vivo microscopic observation as previously described (7, 19). The mesentery was draped over an optically clear viewing pedestal that allowed for transillumination of a 3-cm segment of tissue. The temperature of the pedestal was maintained at 37°C with a constant-temperature circulator (model 80, Fisher Scientific, Pittsburgh, PA). The exposed bowel was draped with saline-soaked gauze, while the remainder of the mesentery was covered with clear plastic wrap (Saran Wrap, Dow Corning, Midland, MI). The exposed mesentery was suffused with warmed bicarbonate-buffered saline (pH 7.4) that was bubbled with 5% CO2-95% N2. The mesenteric preparation was observed through an intravital microscope (Optiphot-2, Nikon, Mississauga, ON, Canada) with a x25 objective lens (Wetzlar L25/0.35, Leitz, Munich, Germany) and a x10 eyepiece. The image of the microcirculatory bed (x1,400 magnification) was recorded using a video camera (Digital 5100, Panasonic, Osaka, Japan) and a video recorder (model NV8950, Panasonic).
Single unbranched mesenteric venules (2540 µm diameter, 250 µm long) were selected for each study. Venular diameter was measured on- or offline using a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX). The number of rolling and adherent leukocytes was determined offline during playback analysis. Rolling leukocytes were defined as white blood cells that moved at a velocity less than that of erythrocytes in a given vessel. The number of rolling leukocytes (flux) was counted using frame-by-frame analysis. To obtain a complete leukocyte rolling velocity profile, the rolling velocity of all leukocytes entering the vessel was measured. A leukocyte was defined as adherent to venular endothelium if it remained stationary for >30 s. Adherent cells were measured at 10-min intervals as described in the experimental protocol and expressed as the number per 100-µm length of venule. Previous work demonstrated that >90% of leukocytes were neutrophils (41).
Microvascular permeability.
The degree of microvascular dysfunction was assessed as vascular albumin leakage in cat mesenteric venules. Briefly, FITC (25 mg/kg iv)-labeled bovine albumin (Sigma, St. Louis, MO) was administered to animals 15 min before the start of the experimental procedure. Fluorescence intensity (420- to 490-nm excitation wavelength, 520-nm emission wavelength) was detected using a silicone-intensified fluorescent camera (model C-2400-08, Hamamatsu Photonics, Hamamatsu, Japan), and images were recorded for playback analysis using a videocassette recorder. The fluorescent intensity of FITC-albumin within a defined area (10 x 50 µm) of the venule under study and in the adjacent perivascular interstitium (20 µm from venule) was measured under control conditions at 60 min of ischemia and at 10 and 60 min of reperfusion. This was accomplished using a video-capture board (Visionplus AT-OFG, Imaging Technology, Bedford, MA) and a computer-assisted digital imaging processor (Optimas, Bioscan, Edmonds, WA). The index of vascular albumin leakage (permeability index) was determined from the ratio of interstitial intensity background to venular intensity background, as previously reported (8, 22).
Experimental protocol.
After a 30-min stabilization period, baseline measurements of blood pressure, SMA blood flow, and vessel diameter were obtained. The preparation was videotaped during a 10-min control period, and then the intestine was exposed to 60 min of ischemia (blood flow 20% of control) and 60 min of reperfusion. In two series of animals (n = 5), an identical protocol was completed; however, 0.5 mg of SOD2 or SOD2/3 was administered via the cannulated jugular vein after 55 min of ischemia. This dose of
1 U/g body wt was chosen on the basis of previous reports that these protocols were efficacious in vivo in other inflammatory models (5, 9, 16, 39).
Neutrophil isolation and cytochrome c reduction assay.
A cytochrome c reduction assay was used to measure the antioxidant capacity of plasma from animals treated with SOD2 or SOD2/3. Human polymorphonuclear leukocytes (PMNs) were used as a source of O2 production. Human neutrophils were harvested from acetate-citrate-dextrose-anticoagulated venous blood collected from healthy donors. All isolation steps were performed at room temperature. Neutrophils were purified by dextran sedimentation (250,000-Da dextran, Spectrum Chemicals) followed by centrifugation through a density gradient (6.07% Ficoll type 400, Sigma) with 10% Hypaque sodium (Winthrop-Breon). Isolated neutrophils were resuspended in Hanks' buffered salt solution and used at a density of 1 x 106 cells/ml. Briefly, PMNs (107/ml) were added to two cuvettes containing PBS with 1.19 mM CaCl2, 0.54 mM MgCl2, and 1.5 mM cytochrome c (Sigma). In the reference sample, SOD1 (from bovine erythrocytes, 264 U/ml; Sigma Chemical) was added, and the reference and experimental samples were read at the same time in a spectrophotometer (model U-2000, Hitachi) at 550 nm. Optical density differences between the two samples were recorded on an online chart recorder (Johns Scientific). After 5 min of baseline measurements, PMA (5 ng/ml; Sigma) was added to both samples, and optical density was recorded for an additional 10 min. In a separate set of experiments, plasma (10 or 50 µl) from SOD2- or SOD2/3-treated animals and PMA (5 ng/ml) were mixed before addition to cuvettes as stated above.
Statistical analysis.
The data were analyzed using standard statistical analysis, i.e., ANOVA and Student's t-test with Bonferroni's correction for multiple comparisons where appropriate. Values are means ± SE. Statistical significance was set at P < 0.05.
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RESULTS
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Changes in mucosal architecture during I/R.
In the small intestine, a single-cell epithelial layer lines the crypts and covers the villi. Changes in mucosal architecture are easily measured by light microscopy. Villus height, mucosal thickness, and crypt depth are the parameters most frequently measured and have been applied in numerous animal and patient studies. Histological examination showed I/R-induced mucosal damage manifested as sloughing of the villus epithelium, reduction in villus height, and infiltration of leukocytes (Fig. 1).

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Fig. 1. Ischemia-reperfusion (I/R)-induced mucosal damage in small intestine. A: ileum from small intestine with normal intestinal architecture (hematoxylin and eosin, x40). B and C: histopathological manifestations of ileum after I/R for 1 and 4 h, respectively. Villus sloughing and profound villus shortening are indicated by arrows in B and C, respectively.
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Hemodynamic factors during I/R.
Table 1 and Fig. 2 summarize hemodynamic factors in animals before and after I/R. As shown in Table 1, the blood pressure in untreated animals did not change during I/R. Similarly, blood pressure in SOD2-treated animals remained approximately equivalent to that determined before ischemia (Table 1). In animals treated with SOD2/3, a small but significant 8% reduction in blood pressure was observed. The blood pressure returned to normal levels within 60 min of reperfusion (Table 1).

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Fig. 2. Superoxide dismutase 2/3 (SOD2/3) reduces intestinal blood flow during I/R. Animals were untreated (A) or treated with SOD2 (B) or SOD2/3 (C) 5 min before reperfusion. Intestinal blood flow was determined before ischemia (cont), during ischemia (isc), and 10, 30, and 60 min after reperfusion. Values are arithmetic means ± SE of 5 animals per group. *P < 0.05 relative to preischemic (cont) level.
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During the ischemic period, intestinal blood flow was reduced by 80% in all animals. Intestinal blood flow in untreated animals returned to 87% of control levels (75 ± 5 ml/100 g) within 10 min of reperfusion (Fig. 2A). At 30 min of reperfusion, blood flow remained at 72 ± 5 ml/100 g but dropped even further after 60 min of reperfusion, with 66 ± 7 ml/100 g blood flow determined in the mesenteric vasculature. In SOD2-treated animals, we observed that intestinal blood flow was also reduced relative to control but did not achieve significance (Fig. 2B). In animals treated with SOD2/3, intestinal blood flow followed a pattern very similar to that of animals not receiving SOD (Fig. 2C).
SOD2/3 inhibits leukocyte recruitment during I/R.
We used intravital microscopy to directly observe leukocyte-endothelial cell interactions in the postcapillary venules of cat mesentery. I/R resulted in a significant increase in the number of rolling leukocytes within the mesentery (Fig. 3). In untreated animals, before I/R we observed 46 ± 7 cells rolling per minute (Fig. 3, untreated, ). Within 30 min of reperfusion, the number of rolling cells in these animals increased almost threefold to 122 ± 25, and by 60 min, this number increased further to 153 ± 20 (Fig. 3, , 30 and 60). In SOD2-treated animals, before I/R we observed 26 ± 4 cells rolling per minute (Fig. 3, SOD2, ). Within 30 min of reperfusion, the number of rolling cells in these animals increased almost threefold to 71 ± 13 (Fig. 3, SOD2, 30). The number of leukocytes rolling remained elevated above control levels over the next 30 min in the SOD2-treated animals but was significantly lower than in the untreated group at 60 min of reperfusion (Fig. 3, SOD2, 60). In animals treated with SOD2/3, we observed 45 ± 6 cells rolling per minute before the ischemic episode (Fig. 3, SOD2/3, ). Over the 60 min of reperfusion, there was a small increase in the number of rolling cells in the mesenteric vasculature, with 64 ± 21 and 60 ± 15 cells/min for 30 and 60 min of reperfusion, respectively (Fig. 3, SOD2/3, 30 and 60). At no time during these particular experiments was there a significant increase in rolling flux, and at 60 min after reperfusion, the number of rolling cells in SOD2/3-treated animals was significantly lower than in the untreated group. These data suggest that O2 is likely partly responsible for the increased rolling during reperfusion.

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Fig. 3. SOD2 and SOD2/3 reduce leukocyte rolling flux during I/R. Animals were untreated or treated with SOD2 or SOD2/3 5 min before reperfusion. Leukocyte rolling flux was determined before ischemia () and 30 and 60 min after reperfusion. Values are arithmetic means ± SE of 5 animals per group. *P < 0.05 relative to preischemic levels; #P < 0.05 relative to levels in untreated animals for the same time point.
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The rolling velocity of leukocytes is an excellent marker of increased cell recruitment and endothelial cell activation (17, 21). In untreated animals, the rolling velocity of the leukocytes in the mesenteric vasculature was reduced significantly during reperfusion. The rolling velocity was reduced from 58 ± 11 to 35 ± 6 and 39 ± 6 µm/s before ischemia and after 30 and 60 min of reperfusion, respectively (Fig. 4, ). In SOD2-treated animals, the rolling velocity of leukocytes in the mesenteric vasculature was also significantly reduced within 30 min of reperfusion: 46 ± 4 vs. 34 ± 5 µm/s before ischemia and at 30 min of reperfusion, respectively (Fig. 4, SOD2, and 30). This reduction in leukocyte velocity was sustained for the 60 min of reperfusion (31 ± 4 µm/s; Fig. 4, SOD2, 60). In contrast to these two groups, the rolling velocity of the leukocytes in the mesenteric vasculature of SOD2/3-treated animals was not significantly reduced from the values observed before the ischemic episode (Fig. 4, SOD2/3).

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Fig. 4. SOD2/3-treated animals exhibit normal leukocyte rolling velocity during I/R. Animals were untreated or treated with SOD2 or SOD2/3 5 min before reperfusion. Leukocyte rolling velocity was determined before ischemia and 30 and 60 min after reperfusion. Values are arithmetic means ± SE of 5 animals per group. *P < 0.05 relative to preischemic levels.
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Because a reduction in rolling velocity causes a retention of rolling leukocytes in blood vessels, we calculated the number of rolling leukocytes in each blood vessel by dividing the rolling flux by the rolling velocity. Using the aforementioned data, we identified the number of rolling leukocytes within a 100-µm length of mesenteric venule for each group of animals. The number of rolling cells per 100-µm length in untreated animals was significantly increased within 30 min of reperfusion (Fig. 5, ). Similarly, the number of rolling cells per 100-µm length in SOD2-treated animals was significantly increased within 30 min of reperfusion (Fig. 5, SOD2, and 30). This increase was sustained for the 60 min of reperfusion in these two groups (Fig. 5, and SOD2, 60). By contrast, administration of SOD2/3 prevented a significant increase in the number of rolling cells per 100-µm length for
60 min of reperfusion (Fig. 5, SOD2/3).

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Fig. 5. SOD2/3-treated animals exhibit a reduced number of rolling cells per vasculature length during I/R. Animals were untreated or treated with SOD2 or SOD2/3 5 min before reperfusion. Number of rolling leukocytes per 100 µm was determined before ischemia and 30 and 60 min after reperfusion. Values are arithmetic means ± SE of 5 animals per group. *P < 0.05 relative to preischemic levels.
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SOD2/3 inhibits leukocyte adhesion and emigration during I/R.
It has previously been shown that neutrophil adhesion and emigration lead to vascular dysfunction (23). I/R induced a significant increase in neutrophil adhesion in postcapillary venules of untreated animals (Fig. 6). In untreated animals, within 30 min of reperfusion, 11 ± 3 adherent cells per 100 µm were observed in the microvasculature, a level that did not change further (Fig. 6, ). Administration of SOD2 to animals before reperfusion neither delayed nor prevented adhesion of leukocytes to the endothelium after the ischemic episode, inasmuch as they exhibited 10 ± 3 adherent cells per 100 µm (Fig. 6, SOD2, and 30). Again no increase in cell adhesion was observed over the next 30 min of reperfusion. By contrast, in the animals treated with SOD2/3, the number of leukocytes adhering did not increase significantly above the 2 ± 1 cells/100 µm observed in the venules before ischemia (Fig. 6, SOD2/3). Cell adhesion in SOD2/3-treated animals was significantly less than that observed in untreated and SOD2-treated animals.

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Fig. 6. SOD2/3-treated animals exhibit reduced leukocyte adhesion during I/R. Animals were untreated or treated with SOD2 or SOD2/3 5 min before reperfusion. Leukocyte adhesion was determined before ischemia and 30 and 60 min after reperfusion. Values are arithmetic means ± SE of 5 animals per group. *P < 0.05 relative to preischemic levels; #P < 0.05 relative to levels in untreated animals for the same time point; P < 0.05 relative to levels in SOD2-treated animals for the same time point.
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The transmigration of leukocytes from the vasculature into the interstitium has been shown to be linked to a heightened inflammatory response (52, 53). We observed that the number of emigrating leukocytes correlated with our cell adhesion and permeability data. In untreated animals, the number of emigrating cells increased from 1.0 ± 0.3 to 5.3 ± 1.0 cells per field of view within 30 min of reperfusion. In these animals, the number of emigrating cells increased further to 8.6 ± 1.7 cells per field of view at 60 min of reperfusion (Fig. 7, ). In SOD2-treated animals, the number of emigrating leukocytes increased from 1.0 ± 0.4 to 6.8 ± 1.4 cells per field of view within 30 min of reperfusion and increased again to 10.4 ± 2.9 within the next 30 min (Fig. 7, SOD2). Administration of SOD2/3 significantly inhibited the number of leukocytes emigrating from the vasculature after I/R. Although we observed a slight increase in the number of leukocytes emigrating from the mesenteric vasculature of these animals, the values were significantly less than those observed in untreated as well as SOD2-treated animals throughout the experiment (Fig. 7, SOD2/3).

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Fig. 7. Leukocyte emigration is inhibited by SOD2/3 during I/R. Animals were untreated or treated with SOD2 or SOD2/3 5 min before reperfusion. Leukocyte emigration was determined before ischemia and 30 and 60 min after reperfusion. Values are arithmetic means ± SE of 5 animals per group. *P < 0.05 relative to preischemic levels; #P < 0.05 relative to levels in untreated animals for the same time point; P < 0.05 relative to levels in SOD2-treated animals for the same time point.
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SOD2/3 inhibits I/R-induced vascular permeability.
Similar results were also observed for vascular permeability responses in these groups of animals. In untreated animals, the vascular permeability increased from 9 ± 2 to 61 ± 12% leakage within 30 min of reperfusion and increased further to 83 ± 13% leakage over the next 30 min of reperfusion (Fig. 8, ). Similarly, in SOD2-treated animals, the vascular permeability increased from 10 ± 2 to 80 ± 10% leakage within 30 min of reperfusion and was sustained (Fig. 8, SOD2). By contrast, administration of SOD2/3 significantly inhibited I/R-induced vascular permeability. Vascular permeability in these animals at 30 min of reperfusion rose to 32 ± 10% leakage (Fig. 8, SOD2/3). At 30 and 60 min of reperfusion, the permeability in SOD2/3-treated animals was significantly less than that observed in untreated and SOD2-treated animals.

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Fig. 8. SOD2/3-treated animals exhibit reduced vascular permeability during I/R. Animals were untreated or treated with SOD2 or SOD2/3 5 min before reperfusion. Intestinal vascular permeability was determined before ischemia as well as 30 and 60 min after reperfusion. Values are arithmetic means ± SE of 5 animals per group. *P < 0.05 relative to preischemic levels; #P < 0.05 relative to levels in untreated animals for the same time point; P < 0.05 relative to levels in SOD2-treated animals for the same time point.
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SOD2/3 remains active and in circulation throughout I/R.
To assess the relative abilities of the administered doses of SOD2 and SOD2/3 to effectively scavenge neutrophil-generated O2· throughout the period of reperfusion, we isolated plasma from animals before ischemia and after 60 min of reperfusion. We used the cytochrome c reduction assay to examine the ability of circulating SOD2 and SOD2/3 to scavenge O2 produced by PMA-stimulated human neutrophils. In the presence of untreated cat plasma (10 µl/ml), PMA-stimulated neutrophils produced 1.2 nM·min1·106 cells1 of O2 (Fig. 9). A similar level of O2 production was detected with the addition of plasma (10 µl/ml) from an SOD2-treated animal 60 min after reperfusion. By contrast, when 50 µl of plasma from the SOD2-treated animal 60 min after reperfusion were added,
35% less detectable O2 was produced. Neutrophils exposed to PMA + plasma (10 µl/ml) from a second animal before I/R produced 1.2 nM·min1·106 cells1 of detectable O2. When 10 µl of plasma from the SOD2/3-treated animal (60 min after reperfusion) were added,
80% less O2 was detected (0.2 nM·min1·106 cells1). These results suggest that SOD2 and SOD2/3 remain in circulation for
65 min after administration but that SOD2/3 is more effective than SOD2 in scavenging neutrophil-generated O2. These results support our previous observation that any SOD bearing a net positive charge at pH 7.4 is dramatically effective in neutrophil-generated O2 because of its electrostatic interaction with the negatively charged cell membrane (36).

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Fig. 9. SOD2/3 is more effective than SOD2 in scavenging PMA-induced O2 production from isolated neutrophils. Isolated neutrophils were resuspended in buffer containing cytochrome c with or without SOD. PMA (5 ng/ml) and plasma (10 or 50 µl) from animals treated with SOD2 or SOD2/3 were added, and optical density was recorded. Results are representative of 1 experiment per group.
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DISCUSSION
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For years there have been many unsuccessful attempts to use SOD as a therapeutic agent to treat ROS-related pathologies. The majority of work has focused on SOD1 and SOD2. However, their pharmacological properties render them poorly suited for therapeutic use. Studies using SOD1 and SOD2 demonstrate that they have the relatively short plasma half-lives of
10 min and 4 h, respectively, depending on species, after intravenous injection with rapid clearance by the kidneys (1, 38). By contrast, SOD3 is an extracellular enzyme known to have a plasma half-life of
20 h in the rabbit (18). More importantly, SOD3 binds to heparin-like proteoglycans on the extracellular membrane, which allows it to directly interact with ROS potentially at exactly the site of O2 production (45). Unfortunately, application of SOD3 to counteract ROS has been thwarted by an inability to mass-produce this enzyme. The generation of the SOD2/3 chimera overcomes all these limitations. The chimeric SOD was genetically engineered to yield the human SOD2 molecule fused to a positively charged COOH terminal tail consisting of a 26-amino acid sequence that comprises the heparin-binding domain of SOD3. This chimeric molecule can now be mass-produced, is stable at physiological pH, and has potent anti-ROS enzymatic activity (9, 38).
It is well recognized that neutrophil infiltration from blood to postischemic tissue is a multistep process that includes initial contact between the neutrophil and the endothelium. Weak transient adhesive interactions then follow to manifest as neutrophil rolling and, ultimately, firm adhesion of the neutrophil to the vessel wall (46, 49). Firm adhesion then allows neutrophils to transmigrate across the vessel wall to final target sites. Functional blocking experiments employing neutralizing antibodies or adhesion molecule-deficient mice implicate P-selectin as the dominant factor for neutrophil rolling in postischemic venules, whereas an interaction between
2-integrins on leukocytes and ICAM-1 on endothelial cells accounts for the firm adhesion (11). Our data would suggest that it is the O2 that may initiate some of those recruitment steps. Indeed, addition of SOD2/3 almost eliminated neutrophil adhesion and emigration. Although there was some attenuation of rolling, it was definitely not complete. This is consistent with our previous work showing a role for oxidant-independent mechanisms including thrombin (43). Although O2 can mobilize the stored pool of P-selectin to the endothelial cell surface, where it then mediates leukocyte rolling, this likely occurs via the formation of H2O2. Because SOD will not reduce H2O2 formation, this may explain the more subtle effect of SOD on rolling than on adhesion or on emigration.
Activated neutrophils produce potent proteases that degrade components of the endothelial basement membrane as well as junction proteins that maintain endothelial barrier function (4). This increase in vascular permeability permits the release of more proinflammatory mediators (e.g., cytokines and chemokines) into and out of the circulation, such that more neutrophils can be recruited to promote inflammation and injury. It stands to reason then that the prevention of excessive neutrophil adhesion and emigration allows the vascular integrity to be maintained. Not only can the emigrated leukocytes release the aforementioned proteases and ROS, their production of cytokines and chemokines can enhance the proinflammatory response in which more leukocytes are recruited to the area for increased necrosis and injury. Clearly, addition of SOD2/3 limits the neutrophil recruitment and, hence, the vascular dysfunction.
Our study supports previous observations in which SOD2/3 exhibited anti-inflammatory properties in models of lung injury, foot edema, and I/R (5, 9, 13, 16, 38). Our work also extends those studies by demonstrating for the first time that SOD2/3 inhibits I/R-induced increased blood pressure, leukocyte recruitment, and vascular permeability. More specifically, our results suggest that administration of SOD2/3 protects at the neutrophil-endothelium interface. From a therapeutic standpoint, similar experiments in which an equivalent amount of SOD2 was administered were not as effective in preventing I/R-induced injury. Although SOD2 administration offered some protection to neutrophil rolling, the resulting number of adhering and emigrating leukocytes was not inhibited. Similarly, the vasculature in SOD2-treated animals exhibited an increase in permeability equivalent to that observed in the untreated I/R animals, and their blood pressure was also elevated. Although previous data have reported excellent results in I/R injury with human SOD1 or SOD2 administration, the experiments required ligation of kidneys to prevent SOD clearance. Clearly, this is not a feasible option therapeutically. By contrast, the antioxidant capacity of plasma from animals treated with SOD2/3 protected against neutrophil activation, despite leaving the kidneys intact. Although the precise mechanism by which SOD2/3 mediates its protective effect is unclear, it is unlikely to involve the regulation of nitric oxide, inasmuch as administration of N-nitro-L-arginine methyl ester before ischemia did not alter the effectiveness of SOD2/3 in our system (unpublished observations). The results here support our contention that O2 is generated at the outer surface of the cell and that SOD can be effective only when positioned within very close proximity of the negatively charged cell surface (36). Taken together, these results suggest that SOD2/3 has much better potential for therapeutic application in I/R-induced injury than mammalian SOD1, SOD2, or SOD3.
The relevance of SOD2/3 combating O2 accumulation and, thereby, indirectly inhibiting leukocyte recruitment and vascular permeability extends beyond localized I/R-induced injury. The release of inflammatory mediators via O2 release into the circulation during I/R is known to activate circulating leukocytes and vascular endothelial cells, enhance the expression of adhesion molecules, and promote leukocyte-endothelial cell interactions in multiple vascular beds. Indeed, a devastating consequence of tissue reperfusion is the development of damage in organs uninvolved in the initial ischemic insult. Multiple organ dysfunction syndrome is the leading cause of death in critically ill patients (37). Multiple organ dysfunction syndrome is a documented consequence of gut (40, 47), liver (25, 50), and skeletal muscle I/R (6) as well as aortic occlusion reperfusion (2, 3). With the administration of SOD2/3, one can prevent neutrophil-endothelial cell interactions at the site of I/R as well as those that occur beyond that local area. SOD2/3 may have applications in other inflammatory conditions. Indeed, a reduction in SOD3 has been noted in numerous diseases. Patients with rheumatoid arthritis exhibit a 50% reduction in SOD activity compared with normal controls, suggesting that reduced SOD activity contributes to endothelial dysfunction in patients with this disease (32). Similarly, SOD3 expression is substantially reduced in patients with coronary artery disease (24). Our data reported here suggest that SOD2/3 has a sufficient half-life to reduce significant neutrophil recruitment and vascular dysfunction. Moreover, SOD2/3 may be useful in any oxidant-dependent inflammatory conditions. Whether SOD2/3 can be administered to alleviate the damaging effects of ROS in a multitude of human pathologies and diseases remains to be investigated.
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GRANTS
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This study was supported by a group grant from the Canadian Institutes of Health Research (to P. Kubes) and by a grant from the Herrick Family Foundation (to J. M. McCord). P. Kubes is a Canada Research Chair and an Alberta Heritage Foundation for Medical Research scientist. C. S. Bonder holds a Canadian Association of Gastroenterology fellowship.
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FOOTNOTES
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Address for reprint requests and other correspondence: P. Kubes, Dept. of Medicine, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1 (E-mail: pkubes{at}ucalgary.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. Section 1734 solely to indicate this fact.
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