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Am J Physiol Heart Circ Physiol 287: H805-H811, 2004; doi:10.1152/ajpheart.00907.2003
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Gene transfer of extracellular superoxide dismutase improves relaxation of aorta after treatment with endotoxin

Donald D. Lund,* Carol A. Gunnett,* Yi Chu, Robert M. Brooks, Frank M. Faraci, and Donald D. Heistad

Departments of Internal Medicine and Pharmacology, Cardiovascular Center, University of Iowa Carver College of Medicine, and Veterans Affairs Medical Center, Iowa City, Iowa 52242

Submitted 25 September 2003 ; accepted in final form 15 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lipopolysaccharide (LPS) impairs vascular function, in part by generation of reactive oxygen species. One goal of this study was to determine whether gene transfer of extracellular SOD (ECSOD) improves vascular responsiveness in LPS-treated rats. A second goal was to determine whether effects of ECSOD are dependent on the heparin-binding domain of the enzyme, which facilitates binding of ECSOD to the outside of cells. Adenoviruses containing ECSOD (AdECSOD), ECSOD with deletion of its heparin-binding domain (AdECSOD-HBD), or a control virus (AdLacZ) were injected intravenously into rats. Three days later, vehicle or LPS (10 mg/kg ip) was injected. After 24 h, vascular reactivity was examined in aortic rings in vitro. Maximum relaxation to acetylcholine was 95 ± 1% (means ± SE) after AdlacZ plus vehicle and 77 ± 3% after AdlacZ plus LPS (P < 0.05). Responses to calcium ionophore A-23187 and submaximal concentrations of nitroprusside also were impaired by LPS. Gene transfer of ECSOD, but not AdECSOD-HBD, improved (P < 0.05) relaxation to acetylcholine and A-23187 after LPS. Maximum relaxation to acetylcholine was 88 ± 3% after LPS plus AdECSOD. Superoxide was increased in aorta after LPS, and the levels were reduced after AdECSOD but not AdECSOD-HBD. LPS-induced adhesion of leukocytes to aortic endothelium was reduced by AdECSOD but not by AdECSOD-HBD. We conclude that after gene transfer in vivo, binding of ECSOD to arteries effectively decreases the numbers of adherent leukocytes and levels of superoxide and improves impaired endothelium-dependent relaxation produced by LPS.

acetylcholine; adenovirus; rats; hydroethidine

LPS PRODUCES IMPAIRMENT OF endothelium-dependent vascular relaxation (3, 4, 15). Several studies suggest that endothelial dysfunction in LPS-treated vessels may be produced, in part, by increased levels of reactive oxygen species (ROS), especially superoxide, in blood vessels (15, 21). SOD, which reduces superoxide to hydrogen peroxide, improves endothelium-dependent relaxation in several pathological conditions in which superoxide is elevated in blood vessels (6, 10, 11, 18–20, 28).

Extracellular SOD (ECSOD), in contrast to other isoforms of SOD, is a secreted protein found predominantly outside of cells (12). A COOH-terminal binding domain has affinity for heparin [thus heparin-binding domain (HBD)] or sulfated proteoglycans, which facilitate binding of ECSOD to the external surfaces of many cells, including endothelial cells (12). Truncated forms of ECSOD, without HBDs or with decreased heparin-binding affinity, normally constitute a small percentage of endogenous ECSOD (12) and may, or may not, be important for vascular function. Decreases in ECSOD with high affinity for heparin, and increases in forms of ECSOD with low affinity for heparin, have been observed in atherosclerosis (12). Thus decreased function of the HBD on ECSOD may play a role in vascular disease.

Like other SODs, ECSOD reduces superoxide in solution, even in the absence of cells. Thus binding is not necessary for enzyme activity, per se. The physical location of ECSOD relative to cells being injured by superoxide, however, may be important in terms of the protection afforded by enzymatic activity. The importance of binding ECSOD to vascular tissue for antioxidant protection of vasomotor function during inflammation is not clear. In recent studies, we suggested that the HBD is necessary for antihypertensive effects of ECSOD (9) and protection against vasospasm after subarachnoid hemorrhage (28). To our knowledge, the present study is the first to examine effects of ECSOD or the role of its HBD on vasomotor function after LPS.

The first goal of this study was to determine whether adenovirus-mediated gene transfer of ECSOD reduces levels of superoxide in blood vessels, attenuates adhesion of leukocytes to aortic endothelium, and improves endothelium-dependent relaxation after LPS. The second goal was to determine whether HBD is required for ECSOD to exert antioxidant effects and improve endothelium-dependent relaxation after LPS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenoviral vector. We used three replication-deficient adenoviruses: 1) a virus was constructed by Dr. Yi Chu, using cDNA from human ECSOD and the cytomegalovirus (CMV) promotor, AdCMVECSOD, which expresses ECSOD (9); 2) AdCMVECSOD-HBD, an ECSOD with deletion of its heparin-binding domain (ECSOD-HBD). Dr. Chu modified the cDNA from human ECSOD to make the recombinant virus (9); and 3) AdCMV LacZ containing the reporter gene for {beta}-galactosidase was used as a control virus (8). Adenovirus was propagated at the Vector Core Laboratory of the University of Iowa and stored at –80°C until used.

Animals. All procedures and handling of animals were reviewed and approved by the institutional Animal Care and Use Committee at the University of Iowa. Adult male Sprague-Dawley rats (300–400 g) were anesthetized with pentobarbital sodium (50 mg/kg ip), and adenovirus (0.25 ml of 1 x 1012 particles/ml in 3% sucrose in PBS) was injected intravenously. Three days after viral injections, rats were treated with vehicle or LPS (Escherichia coli stereotype 0128:B12, 10 mg/kg ip). Twenty-four hours later, rats were euthanized by an injection of pentobarbital sodium (150 mg/kg ip) followed by exsanguination. Experiments with AdECSOD and AdECSOD-HBD were performed independently with separate control and LPS ± Lac Z animals in each experiment.

The aorta was quickly removed and placed in cold (4°C) oxygenated Krebs solution (in mM: 133 NaCl, 4.7 KCl, 1.35 NaH2PO4, 16.3 NaHCO3, 0.61 MgSO4, 7.8 glucose, and 2.52 CaCl2). Aortas were cut into rings (3–4 mm in length). Thoracic aortas were used in all studies. Sections from a single aorta were used for vascular function studies and either lucigenin or histochemical studies. Each vessel was cut into three segments 3–4 mm in length to be used for ring studies. We used two additional segments 2.5–3 mm in length for histochemical or lucigenin measurements. All procedures and handling of animals were reviewed and approved by the institutional Animal Care and Use Committee at the University of Iowa.

Detection of superoxide. Hydroethidine, an oxidative fluorescent dye, was used to evaluate levels of superoxide in aorta in situ, as described previously (15, 23). Cells are permeable to hydroethidine, and, in the presence of superoxide, hydroethidine is oxidized to fluorescent ethidium bromide and is trapped by intercalation with DNA. Unfixed frozen ring segments were cut into 30-mm sections and placed on glass slides. Hydroethidine (2 x 10–6 M) was applied topically to each tissue section and cover slipped. Slides were incubated in a light-protected, humidified chamber at 37°C for 30 min. Images were obtained with a laser-scanning, confocal microscope equipped with a krypton-argon laser. Aorta from normal and LPS-treated rats were processed and imaged in parallel.

Superoxide levels were also measured by lucigenin-enhanced chemiluminescence as described previously (23, 24). Vessel segments were placed in 0.5 ml PBS and lucigenin 5 µM, and relative light units were measured for 5 min. Background counts were determined and subtracted and relative light units were normalized to surface areas.

Vasomotor responses. Aortic rings were mounted on stainless-steel hooks at optimal resting tension (2 g) in individual organ baths in Krebs bicarbonate solution at 37°C and aerated with 95% O2-5% CO2. Tension was periodically adjusted to the desired level during a 45-min equilibration period. After initial suspension of vascular rings, contraction of vessels increases with the first two or three exposures to contractile stimuli. KCl is used in these experiments to prepare vessels for reproducible dose-response curves to other vasoactive agents. Vascular rings were contracted twice with KCl (100 mM), and rinsed three times after each contraction. Responses to phenylephrine (10–9 to 10–5 M) were then examined.

Responses to endothelium-dependent dilators, acetylcholine (10–9 to 10–5 M), A-23187 (10–9 to 10–5 M), and the endothelium-independent dilator sodium nitroprusside (10–9 to 10–5 M) were examined after precontraction of vessels with the EC50 dose of phenylephrine. Contractile responses are expressed as grams of tension, and relaxation is expressed as percent of contraction produced by EC50 dose of phenylephrine.

Adhesion of leukocytes. Rings of aorta were pinned out flat, fixed in formalin, and stained with Wrights stain for histological evaluation. Vessel segments were examined en face with a dissecting light microscope. In randomly selected fields, the number of leukocytes in a test grid was counted. Six cross-sectional fields were counted in each rat and one average value was calculated and reported as the number of leukocytes/mm2 of aorta.

Drugs. LPS, acetylcholine chloride, L-phenylephrine hydrochloride, sodium nitroprusside, and lucigenin were dissolved in normal saline. Hydroethidine was obtained from Molecular Probes and suspended in dimethyl sulfoxide at a concentration of 10–2 M.

Statistical analysis. All data are expressed as means ± SE. Intergroup comparisons were performed by using one-way ANOVA to test for difference among treatment groups, followed by Bonferroni's corrected t-test. Comparisons between LPS-treated and normal groups were made by using Student's paired t-test. Differences were considered to be significant when P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Superoxide levels. Levels of superoxide (hydroethidine fluorescence) were increased in aorta from rats given LPS (Fig. 1B). The increase in superoxide was observed primarily in endothelium and adventitia. In aortas from rats given LPS and treated with AdECSOD, hydroethidine fluorescence was less than in rats that received LPS alone. In rats given LPS, fluorescence in aortas was similar in rats treated with AdECSOD-HBD or AdLacZ.



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  		Fig. 1. Superoxide in the aorta detected with hydroethidine. Aorta becomes fluorescent red in the presence of superoxide. Confocal fluorescent photomicrographs of sections of aorta control (A), LPS-treated + AdLacZ (B), and LPS-treated + adenoviruses containing extracellular SOD (AdECSOD) (C). Aorta from rats treated with LPS + AdLacZ had increased levels of superoxide in the endothelium and adventitia, and LPS-treated + AdECSOD tended to reduce superoxide (n = 4 rats).

 
On the basis of measurement of lucigenin chemiluminescence, superoxide levels were higher in aortas from rats given LPS than in normal, control aortas (Fig. 2A). Superoxide levels in aortas from rats given LPS and treated with AdECSOD were lower than in rats treated with LPS alone. Superoxide in aorta from rats treated with LPS plus AdECSOD-HBD was not different from control (Fig. 2B).



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  		Fig. 2. A: superoxide levels (lucigenin) in rats treated with vehicle (control), LPS + AdLacZ, and LPS + AdECSOD. Values are means ± SE (n = 8 rats in each group). *P ≤ 0.05 vs. control. B: superoxide levels (lucigenin) in rats treated with vehicle (control), LPS + AdLacZ, and LPS + AdECSOD-heparin-binding domain (HBD). Values are means ± SE (n = 7 rats in each group). *P ≤ 0.05 vs. control. RLU, relative light units.

 
Vasomotor responses. In aortic rings from rats given LPS, phenylephrine produced less contraction than in control rats (Fig. 3). Response to phenylephrine in rats given LPS was not altered by transfection with ECSOD (Fig. 3) or AdECSOD-HBD (data not shown).



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  		Fig. 3. Contraction of aorta to phenylephrine in rats treated with vehicle (control), LPS + AdLacZ, and LPS + AdECSOD. EC50 = 189 ± 35 nM (control), 551 ± 27 nM (LPS ± AdlacZ), and 385 ± 97 nM (LPS ± AdECSOD). Values are means ± SE (n = 8 rats in each group). *P ≤ 0.05 vs. control.

 
In rings from rats given LPS, maximal relaxation to acetylcholine was significantly less (77 ± 3%) than normal (96 ± 1%) rats (Fig. 4A). Relaxation to acetylcholine was augmented in vessels from rats given LPS and transfected with AdECSOD (88 ± 3%), compared with AdLacZ (Fig. 4A). In rats given LPS, AdECSOD-HBD did not improve responses to acetylcholine (Fig. 4B).



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  		Fig. 4. A: relaxation of aorta to acetylcholine in rats treated with vehicle (control), LPS + AdLacZ, and LPS + AdECSOD. EC50 = 33.5 ± 3.4 nM (control), 104 ± 25 nM (LPS ± AdlacZ), and 31.8 ± 5.5 nM (LPS ± AdECSOD). Values are means ± SE (n = 8 rats in each group). *P ≤ 0.05 vs. control; +P ≤ 0.05 vs. LPS-AdECSOD. EC50 concentrations for phenylephrine were used to preconstrict arteries for relaxation curves. Maximum preconstriction was 2.3 ± 0.4 g in control vessels, 1.4 ± 0.2 g in LPS-AdLacZ vessels, and 1.1 ± 0.2 g in LPS-AdECSOD vessels. B: relaxation of aorta to acetylcholine after vehicle (control), LPS + AdLacZ, and LPS + AdECSOD-HBD. EC50 = 383 ± 40 nM (control), 109 ± 50 nM (LPS ± AdlacZ), and 179 ± 40 nM (LPS ± AdECSOD-HBD). Values are means ± SE (n = 7) in each group. *P ≤ 0.05 vs. control. Maximum preconstriction was 2.3 ± 0.1 g in control vessels, 1.4 ± 0.2 g in LPS-AdLacZ vessels, and 1.4 ± 0.2 g in LPS-AdECSOD-HBD vessels.

 
Responses to calcium ionophore A-23187, an endothelium-dependent vasodilator, were significantly less in rats given LPS than control rats (Fig. 5A). Transfection with ECSOD in rats given LPS improved relaxation to A-23187.



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  		Fig. 5. A: relaxation of aorta to A-23187 in rats treated with vehicle (control), LPS + AdLacZ, and LPS + AdECSOD. EC50 = 30.8 ± 3.9 nM (control), 32.5 ± 21.4 nM (LPS ± AdlacZ), and 26.6 ± 5.5 nM (LPS ± AdECSOD). Values are means ± SE (n = 7 in each group). *P ≤ 0.05 vs. control. +P ≤ 0.05 vs. LPS-AdECSOD. Maximum preconstriction was 1.6 ± 0.3 g in control vessels, 1.3 ± 0.3 g in LPS-AdLacZ vessels, and 1.1 ± 0.2 g in LPS-AdECSOD vessels. B: relaxation of aorta to sodium nitroprusside after LPS. EC50 = 5.69 ± 0.13 nM (control), 33.4 ± 11.1 nM (LPS ± AdlacZ), and 18.7 ± 5.3 nM (LPS ± AdECSOD). Values are means ± SE (n = 8). *P ≤ 0.05 vs. control. Maximum preconstriction was 2.4 ± 0.3 g in control vessels, 1.5 ± 0.2 g in LPS-AdLacZ vessels, and 1.1 ± 0.2 g in LPS-AdECSOD vessels.

 
Submaximal responses to sodium nitroprusside, an endothelium-independent vasodilator, were attenuated in rats given LPS, but maximal responses were not altered (Fig. 5B). Transfection with ECSOD did not alter the response to nitroprusside in rats given LPS (Fig. 5B). After AdECSOD-HBD, responses to nitroprusside were similar to those of AdECSOD (data not shown).

Leukocyte adhesion. The number of leukocytes bound to the aorta was greater in rats given LPS than in normal aortas (Fig. 6A). Adhesion of leukocytes in rats given LPS and treated with AdECSOD was less than in rats treated with LPS and AdLacZ. Leukocytes in aorta from rats given LPS and treated with AdECSOD-HBD were not different from rats treated with LPS and AdLacZ (Fig. 6B).



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  		Fig. 6. A: adhesion of leukocytes to the aorta in rats treated with vehicle (control), LPS + AdLacZ, and LPS + AdECSOD. Values are means ± SE (n = 7). *P ≤ 0.05 vs. control. B: adhesion of leukocytes to the aorta in rats treated with vehicle (control), LPS + AdLacZ, and LPS + AdECSOD-HBD. Values are means ± SE (n = 7). *P ≤ 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of this study are the following: 1) levels of superoxide elevated in the aorta after LPS and decreased toward normal by gene transfer of ECSOD; 2) relaxation to acetylcholine and A-23187 is impaired in aorta from LPS-treated rats and is improved by gene transfer of ECSOD, but impaired contraction after LPS is not improved by ECSOD; 3) in rats given LPS, there is increased adhesion of leukocytes to the aorta and adhesion is decreased by gene transfer of ECSOD; and 4) gene transfer of modified ECSOD that cannot bind to external cell surfaces (ECSOD-HBD) does not improve vasomotor responses, or attenuate leukocyte adhesion after LPS.

Superoxide levels. Elevated levels of ROS are thought to contribute to vasomotor dysfunction during inflammation (4, 15, 26). Previous studies (4, 15, 20) suggest that levels of ROS, including superoxide, are elevated in blood vessels after LPS. Pharmacological reduction of superoxide in arteries improves endothelium-dependent relaxation after LPS (15), but at least one study suggests that administration of CuZnSOD and SOD-mimetics after LPS does not improve endothelium-dependent relaxation in rat aorta (4). Thus the importance of elevated levels of superoxide and the role of SOD in vascular dysfunction after LPS is not clear. This is the first study to examine effects of ECSOD on vascular dysfunction produced by LPS.

Data in the present study suggest that levels of superoxide are elevated in aorta after LPS, and the increase in superoxide occurs primarily in endothelium and to a lesser extent in adventitia. Previously, we found by immunostaining that ECSOD protein was observed bound to endothelium in aorta and carotid arteries after administration of AdECSOD but not AdECSOD-HBD (9). These results support the hypothesis that the HBD is necessary for binding of ECSOD to blood vessels. In the present study, AdECSOD, but not AdECSOD-HBD, effectively reduced levels of superoxide in arteries from LPS-treated rats. Thus HBD appears to be important for reduction of levels of superoxide in arteries. Superoxide binds with great affinity to NO, and elevated levels of superoxide in the extracellular space may effectively decrease transcellular passage of NO between endothelium and vascular muscle (25). Thus one likely mechanism by which bound ECSOD improves vasorelaxation is by increasing bioavailability of endothelium-derived NO to relax vascular muscle.

Vasoconstriction. The finding that vasoconstriction is impaired after LPS is consistent with several previous studies (2, 3, 13, 14, 27). The finding that ECSOD did not improve impaired constrictor responses to phenylephrine suggests that extracellular superoxide may not be functionally important in impairment. LPS induces inducible nitric oxide (NO) synthase (iNOS) in arteries, and iNOS mediates impaired constrictor responses produced by LPS (3, 14, 27). A likely mechanism by which iNOS impairs vasoconstrictor responses is by a direct effect of high levels of NO. Because impairment of vasoconstriction is mediated by NO and not superoxide, it is not surprising that ECSOD did not improve constrictor responses.

Vasorelaxation. Impaired relaxation of arteries to acetylcholine after LPS is consistent with data from previous studies (4, 15). Some studies suggest that ROS are elevated in arteries after LPS (5, 15, 20), but at least one study suggests that exogenous administration of SOD or SOD-mimetics does not improve relaxation (4). Current data with AdECSOD suggest that superoxide is an important mediator of impaired endothelium-dependent relaxation after LPS. One explanation for the difference in the present study and that of Brandes et al. (4) is that ECSOD is extracellular, whereas the primary effect of CuZnSOD and SOD-mimetics is intracellular. Our data suggest that ECSOD can protect against vascular dysfunction during inflammation.

A-23187 is a calcium ionophore that activates calcium-dependent endothelial NOS by directly increasing intracellular calcium levels by a mechanism that is not mediated by membrane receptors. Impaired responses to A-23187, in addition to impaired responses of acetylcholine, suggest that impairment is not limited to muscarinic receptors and subsequent signal transduction. Thus after LPS, superoxide exerts its inhibitory effects on NO, generated by endothelium, and its effector functions.

Although reduction of superoxide is accomplished by both ECSOD and ECSOD-HBD proteins in plasma (9), binding of ECSOD to blood vessels appears to be necessary to improve endothelium-dependent relaxation. In a previous study (9) using gene transfer in vivo in rats, ECSOD activity was elevated in plasma after either AdECSOD or AdECSOD-HBD. Based on immunohistochemistry studies, however, ECSOD protein was found bound to endothelium and vascular muscle only after gene transfer of AdECSOD but not AdECSOD-HBD (9). Thus the HBD is critical for localization of ECSOD to the vascular wall. It is interesting to speculate that in an intact system in vivo, unbound ECSOD-HBD in plasma might have protective effects on endothelial function. In the same previous study, however, vascular resistance in rats was changed only by ECSOD and not ECSOD-HBD. In another study (28), injection of AdECSOD into cerebrospinal fluid produced elevated concentrations of ECSOD in basilar arteries and protected against vasospasm after subarachnoid hemorrhage. In contrast, AdECSOD-HBD increased ECSOD in cerebrospinal fluid, but not in arteries, and failed to improve vasospasm. Our vascular function and superoxide data also are consistent with findings of Abrahamsson et. al (1), who reported that ECSOD (with binding capacity) but not CuZnSOD (without binding capacity), preserved vasorelaxation in the presence of exogenous superoxide, and that protective effects were reversed by the addition of heparin, a competitor for ECSOD binding sites. Thus, consistent with earlier studies in which binding of ECSOD to arteries was important for protective effects on vascular function, we now demonstrate that after gene transfer in vivo, binding of ECSOD to arteries via the HBD is necessary to improve endothelium-dependent relaxation after treatment with LPS.

Data with AdECSOD-HBD suggest that binding of ECSOD to vascular cells is critical for protective effects of the antioxidant enzyme. Binding of ECSOD to endothelium after gene transfer may improve endothelial function by direct effects on vascular cells and/or by indirect effects relating to decreased intravascular adhesion of leukocytes. To our knowledge, the present study is the first to demonstrate that ECSOD with normal binding capacity, but not ECSOD-HBD, reduces numbers of adherent leukocytes in blood vessels after LPS. The role of the HBD in ECSOD may be of clinical importance because an R213G polymorphism within the HBD has been described in humans (12). Subjects with the polymorphism have 8- to 10-fold increased activity of ECSOD in serum but decreased affinity of ECSOD for heparin and tissues (12). We speculate that subjects with this gene variant may have increased susceptibility to endotoxin. Results of gene transfer in the present study suggest that in vivo administration of ECSOD with fully active binding capacity may afford protection against vascular dysfunction produced by endotoxin. Based on effects of gene transfer of ECSOD in previous studies (9, 12) of hypertension in rats and subarachnoid hemorrhage in rabbits and our present studies, we think it is likely that administration of exogenous SOD, that can bind to blood vessels, may prove to be beneficial in settings other than acute inflammation, such as atherosclerosis and diabetes, in which vascular superoxide is elevated.

Normal maximum relaxation to nitroprusside suggests that impaired responses to acetylcholine were not due to generalized damage to aorta produced by LPS or other interventions. The finding that ECSOD improved responses to acetylcholine after LPS provides additional evidence that altered vasomotor responses were not due primarily to irreversible damage to vessels.

Decreased sensitivity to submaximal concentrations of nitroprusside, which was only partially reversed by ECSOD, suggests that extracellular superoxide may not be the only mediator of deleterious effects of LPS on vasorelaxation. ECSOD substantially improved responses to acetylcholine but did not completely restore them to normal. The ECSOD-insensitive portion of impaired responses to acetylcholine may reflect an additional mechanism(s) of dysfunction that is consistent with effects on nitroprusside. Data with ECSOD do not rule out a role for intracellular superoxide in impaired NO-mediated relaxation. Effects of ECSOD on responses to acetylcholine, however, strongly suggest that extracellular superoxide plays a major role in impairment of endothelium-dependent relaxation in this model.

We also examined adhesion of leukocytes in arteries after LPS. LPS increased numbers of leukocytes adherent to the aorta. AdECSOD, but not AdECSOD-HBD, decreased the number of adherent leukocytes after LPS. Although mechanisms by which AdECSOD reduced leukocyte adhesion were not examined in the present study, we speculate that adhesion molecules and/or chemokines may be involved. Previous studies (7, 29) using tissue culture suggest that superoxide may enhance and antioxidants may decrease expression of vascular adhesion molecules in endothelial cells.

Data in the present study do not allow us to determine whether the mechanism by which ECSOD improves vasorelaxation depends more on decreased numbers of leukocytes or on decreased superoxide from vascular sources. Leukocytes produce ROS, including superoxide, and may contribute to elevated levels of oxidants in endothelium during inflammation (21, 26). It is not clear, however, whether superoxide from vascular cells or leukocytes accounts for impairment of endothelial function. Proinflammatory cytokines activate oxidases (17, 22). Because leukocytes are important sources of proinflammatory cytokines, they may contribute to vascular dysfunction during inflammation by activation of oxidases within vascular cells. In a previous study, data suggested that arteries incubated with LPS in vitro with no potential for leukocyte recruitment have impaired endothelium-dependent relaxation (16). Whether or not superoxide that impairs endothelial function after LPS originates in leukocytes or vascular cells, our data suggest that ECSOD has important anti-inflammatory effects by reducing numbers of leukocytes on endothelium.

In conclusion, endothelium-dependent vasorelaxation is impaired by LPS and restored by gene transfer of ECSOD. Because ECSOD is an extracellular enzyme (12), extracellular superoxide appears to be a key mediator of vascular dysfunction after LPS. Data in this study demonstrate protection of endothelium-dependent relaxation in arteries during inflammation by anti-inflammatory and antioxidant effects of ECSOD. Furthermore, data from studies with ECSOD-HBD provide evidence that binding of ECSOD to vascular tissue is necessary for protective effects. We speculate that SOD that binds to extracellular matrix has therapeutic potential for treatment of vascular dysfunction associated with inflammation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-16066, HL-62984, NS-24621, HL-38901, and DK-54759 and funds from the Department of Veterans Affairs and the Carver Trust Research Program of Excellence of the University of Iowa.


   ACKNOWLEDGMENTS
 
We thank Pam Tompkins for technical assistance and Arlinda LaRose for secretarial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. D. Heistad, Dept. of Internal Medicine, Univ. of Iowa, 200 Hawkins Dr., Iowa City, IA 52242 (E-mail: carol-gunnett{at}uiowa.edu).

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.

* D. D. Lund and C. A. Gunnett contributed equally to this study. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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