Gene Transfer of Endothelial Nitric Oxide Synthase Reduces Angiotensin II–Induced Endothelial Dysfunction
Abstract—Angiotensin II stimulates vascular NADPH oxidase to produce superoxide, which can react with nitric oxide and impair vasomotor function. We tested the hypothesis that the overexpression of endothelial nitric oxide synthase (eNOS) or superoxide dismutase (SOD) would correct angiotensin II–induced endothelial dysfunction. We examined the effects of the gene transfer of eNOS or 2 isoforms of SOD to the aorta in angiotensin II–treated rabbits on vasomotor function. New Zealand White rabbits were treated for 1 week with angiotensin II (100 ng · kg−1 · min−1) or saline by osmotic minipumps. In angiotensin II–treated rabbits, mean blood pressure was 107±8 mm Hg; it was 67±5 mm Hg in saline-infused rabbits (P<0.05). In aortas from angiotensin II–treated rabbits, lucigenin-enhanced chemiluminescence demonstrated a 2.5-fold increase in superoxide levels, and the oxidative fluorescent probe hydroethidine indicated increased superoxide levels throughout the vascular wall, especially in the endothelium and adventitia. Maximal relaxation to acetylcholine was less in aortas from rabbits treated with angiotensin II (72±5% versus 87±4% in saline-treated rabbits; P<0.01), but responses to sodium nitroprusside were similar. Segments of the thoracic aorta were incubated in vitro with an adenoviral vector that expressed eNOS, copper zinc SOD (CuZnSOD), extracellular SOD (ECSOD), or β-galactosidase. β-Gal treatment with adenovirus containing the gene for eNOS (AdeNOS) but not adenovirus containing the gene for β-gal (Adβ-gal) (control virus) restored responses to acetylcholine (82±3% after AdeNOS and 67±4% after Adβ-gal). Gene transfer of CuZnSOD or ECSOD did not improve the endothelium-dependent relaxation of the aorta in rabbits that received angiotensin II. Thus, gene transfer of eNOS, but not SOD, effectively restores vasomotor function in angiotensin II–infused rabbits.
Impairment of the endothelium-derived nitric oxide (NO) system in a variety of disease states has been attributed to the reduced generation of NO from endothelial NO synthase (eNOS)1 or the inactivation of NO by superoxides.2 3 Several reports indicate that the administration of antioxidant compounds, such as superoxide dismutase (SOD), ascorbic acid, glutathione, or vitamin C, decreases blood pressure and restores the response to acetylcholine or carbachol in hypertensive patients4 and in spontaneously hypertensive rats.5
In atherosclerosis6 and some forms of hypertension, levels of angiotensin II (Ang II) may be increased. Ang II reportedly impairs vascular relaxation, in part, by increasing the production of superoxide anion via a membrane-bound NADPH oxidase.7 In Ang II–induced hypertension in rats, the impairment of vasodilator responses to acetylcholine and calcium ionophore is improved by treatment with liposome-encapsulated SOD.8 On the basis of these observations, we hypothesized that the gene transfer of SOD to vessels after treatment with Ang II would improve vascular relaxation in a manner similar to the effects of SOD in normal vessels.9
In previous studies, the generation of superoxide by Ang II in blood vessels was measured using lucigenin-enhanced chemiluminescence.8 10 Because lucigenin primarily detects superoxide from the endothelium and adventitia,11 it has been assumed that these tissues are the source of the superoxide in Ang II–treated animals. In cell culture, however, Ang II also increases the smooth muscle cell generation of superoxide.7 In this study, we used hydroethidine to test the hypothesis that Ang II may also increase superoxide levels in the smooth muscle cells of vessels from Ang II–treated animals.
If superoxide levels are elevated in vessels after Ang II treatment, the gene transfer of eNOS might not improve NO-mediated dilation, because superoxide can react with NO to produce the potent oxidant peroxynitrite.12 However, because NO avidly binds to superoxide,13 the overexpression of NO through the gene transfer of eNOS might augment relaxation by increasing the bioavailability of NO. Therefore, we also examined the effects of the gene transfer of eNOS in Ang II–induced vasomotor dysfunction to test the hypothesis that increases in eNOS would be sufficient to augment vascular relaxation.
Adult male New Zealand White rabbits (2.0 to 3.4 kg; n=33) were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg). Using sterile techniques, an incision was made in the midscapular region, and osmotic minipumps (Alzet model 2 ML2, Alza Corp) containing Ang II (infusion rate, 100 ng · kg−1 · min−1) or saline were implanted. In preliminary experiments, rabbits that received a higher dose (500 ng · kg−1 · min−1) of Ang II appeared unhealthy, with weight loss and a paradoxical decrease in blood pressure.
Four different recombinant adenoviruses were used for gene transfer. (1) a recombinant adenovirus driven by a cytomegalovirus (cmv) promoter (AdCMVβ-gal) carried the reporter gene for β-galactosidase and was used as a control virus; (2) AdCMVCuZnSOD (a gift from Dr John Engelhardt, University of Iowa, Iowa City) contained cDNA for human copper zinc SOD (CuZnSOD); (3) AdCMVECSOD carried cDNA for human extracellular SOD (a gift from Dr James D. Crapo, National Jewish Medical and Research Center, Denver, Colo); and (4) AdCMVeNOS carried cDNA for bovine eNOS (a gift from Dr David Harrison, Emory University, Atlanta, Ga). The adenoviral vectors were constructed by the University of Iowa Gene Transfer Vector Core using a method described previously.14 PBS containing 3% sucrose was used as a virus-free control.
Transduction of Arteries
After 7 days of infusion with Ang II or saline, the rabbits were anesthetized, and blood pressure was measured. The rabbits were killed by an overdose of pentobarbital, and the thoracic aorta was removed. Vessels were placed in cold Krebs bicarbonate solution with the following composition (in mmol/L): NaCl 118, KCl 4.7, KH2PO4 1.2, MgSO47H2O 1.2, d-glucose 11.1, NaHCO3 25.0, and CaCl2H2O 2.54. Loose connective tissue was removed, and the vessels were cut into rings 3 mm in length.
Segments of the thoracic aorta were incubated at 37°C in 150 μL of viral suspension containing 1010 plaque-forming units/mL of virus (either AdCMVβ-gal, AdCMVCuZnSOD, AdCMVECSOD, or AdCMVeNOS) or vehicle. After 2 hours, viral suspension or vehicle was removed. Rings were rinsed with PBS and incubated with minimum essential medium containing 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C. The gas in the incubator was 5% CO2 and 95% room air. We did not bubble oxygen through the solution at the time of transduction or subsequent incubation. After 24 hours, rings were removed and used for oxidative fluorescent staining, the detection of superoxide by lucigenin-enhanced chemiluminescence, or the recording of isometric tension in the organ chambers.
Detection of Superoxide
Production of superoxide in aortas was measured by the response to lucigenin-enhanced chemiluminescence, as described previously.15 Vascular rings were placed in a polypropylene tube containing 1 mL PBS and lucigenin (250 μmol/L in 14 samples and 5 μmol/L in 3 samples). The tube was placed in a Monolight 2010 luminometer. The luminometer reported relative light units emitted, which were integrated over 5 minutes. Dark current readings (photomultiplier background signal) were automatically subtracted. Background counts, which were determined from vessel-free preparations, were subtracted from the readings obtained with vessels. In some rings, superoxide generation was measured after vessels were preincubated with polyethylene-glycolated (PEG) SOD (250 U/mL) for 30 minutes. The surface area of the lumen was imaged with a video camera; it was calculated with National Institutes of Health image software for each vascular segment to normalize superoxide levels.
Oxidative Fluorescent Microtopography
The oxidative fluorescent dye hydroethidine was used to evaluate the in situ concentration of superoxide, as described previously.11 Hydroethidine is freely permeable to cells and, in the presence of superoxide, is oxidized to ethidium bromide (EtBr), where it is trapped by intercalation with DNA. EtBr is excited at 488 nm and has an emission spectrum of 610 nm. In cell-free assays, hydrogen peroxide, NO, and peroxynitrite do not react with hydroethidine to increase EtBr fluorescence. Unfixed frozen rings of aortic segments were cut into 30-μm-thick sections and placed on a glass slide. Hydroethidine (2×10−6 mol/L) was topically applied to each tissue section and coverslipped. Slides were incubated in a light-protected, humidified chamber at 37°C for 30 minutes. Images were obtained with a Bio-Rad MRC-1024 laser scanning confocal microscope equipped with a krypton/argon laser. Laser settings were identical for the acquisition of images from all samples. Fluorescence was detected with a 585-nm long-pass filter. Sections were subsequently stained with nuclear fast red. First, images of aortas from rabbits that were treated with saline were measured. After adjusting the basal settings of the confocal microscope, images of aortas from rabbits that were infused with Ang II were measured.
Aortic rings were suspended in organ chambers (bubbled with 95% O2 and 5% CO2 at 37°C) containing Krebs bicarbonate solution to measure isometric tension. Rings were progressively stretched to 8g of resting tension, which was determined by repeated stimulation with KCl (100 mmol/L) to be the optimal tension for the vessels. Vessels were allowed to equilibrate for 30 minutes; then, they were contracted twice with 100 mmol/L KCl.
After the vessels were rinsed, their responses to phenylephrine, the endothelium-dependent dilator acetylcholine, or the endothelium-independent dilator sodium nitroprusside were determined. In some rings, responses to acetylcholine were also determined after a 30-minute incubation in Nω-nitro-L-arginine (L-NNA, 10−4 mol/L), an inhibitor of NO synthase, or a 60-minute incubation in PEG-SOD (100 U/mL).
Results are expressed as mean±SE. Relaxations are the percent change from the precontracted tension. Data were analyzed with a 1 way ANOVA with post hoc Bonferroni correction.
Acetylcholine chloride, L-phenylephrine hydrochloride, sodium nitroprusside, L-NNA, PEG-SOD, and lucigenin were obtained from Sigma and dissolved in PBS. Hydroethidine was obtained from Molecular Probes, Inc, and suspended in dimethyl sulfoxide at a concentration of 10−2 mol/L, where it was stored in aliquots at −80°C until use. Subsequent dilution was performed in PBS.
Mean arterial blood pressure was significantly higher in rabbits that received Ang II (107±8 mm Hg, n=23; P<0.05) than in rabbits that received saline (67±5 mm Hg, n=10).
Vascular Superoxide Levels After Infusion of Ang II
We used 2 methods to detect superoxide. Levels of superoxide detected with lucigenin were 2.5 times higher in vessels from rabbits that received an infusion of Ang II (n=9) than those that received saline (n=4) (28±11 versus 11±3 relative light units per minute per area; P<0.05). Addition of exogenous PEG-SOD (250 U/mL) (n=5) significantly reduced superoxide levels in vessels from rabbits that received Ang II (Figure 1⇓).
After 24 hours of incubation in minimum essential medium at 37°C, superoxide levels in vessels from rabbits treated with Ang II (n=4) were 1.7-fold higher than those in control animals (n=4). When measured with a low concentration of lucigenin (5 μmol/L) to avoid the potential autoxidation of lucigenin,16 17 superoxide levels were also increased 1.7-fold in vessels from rabbits treated with Ang II (n=3) compared with controls.
After incubation with hydroethidine, a marked increase in EtBr fluorescence occurred in the aortas from rabbits that received Ang II, which reflected an increase in superoxide (n=3). EtBr fluorescence was most prominent in the endothelium and adventitia, but it was also detected in smooth muscle cells (Figure 2⇓).
Effects of Gene Transfer of eNOS, CuZnSOD, and Extracellular SOD on Vascular Responses
Contractile responses to phenylephrine tended to be enhanced in the aortas from rabbits that received Ang II, but these differences were not significant when compared with control rabbits (Figure 3A⇓). The gene transfer of β-gal or eNOS did not alter the contraction to phenylephrine (Figure 3A⇓).
Maximal relaxation to acetylcholine in aortas from rabbits that received Ang II was impaired compared with vessels from rabbits that received saline (72±5% in Ang II–treated animals versus 87±4% in controls, P<0.01; EC50, 6.77±0.07 log(mol/L) in Ang II–treated animals versus 7.17±0.15 in controls, P<0.05; Figure 3B⇑). Gene transfer of eNOS, but not β-gal, normalized relaxation to acetylcholine in aortas from Ang II–treated rabbits (82±3% in Ang II–treated rabbits versus 7.25±0.08 in controls for eNOS; 67±4% in Ang II–treated rabbits [P<0.05 versus eNOS] versus 6.82±0.04 in controls [P<0.05 versus eNOS] for β-gal; Figure 3B⇑).
L-NNA abolished responses to acetylcholine before and after the gene transfer of eNOS (Figure 3B⇑). Relaxation in response to sodium nitroprusside was similar in rabbits that were infused with saline or Ang II and treated with adenovirus containing the gene for β-gal (Adβ-gal), adenovirus containing the gene for eNOS (AdeNOS), and vehicle (Figure 3C⇑).
Gene transfer of CuZnSOD or extracellular SOD (ECSOD) to aortas from rabbits that received Ang II had no effect on responses to acetylcholine (maximal response, 73±6%; EC50, 6.90±0.10 for CuZnSOD [Figure 4A⇓] and maximal response, 66±8%; EC50, 6.80±0.12 for ECSOD [Figure 4B⇓]).
PEG-SOD augmented the response to acetylcholine in aortas from rabbits that received Ang II (Figure 5⇓). The effects of PEG-SOD treatment were smaller than those of AdeNOS.
In this study, we demonstrated that (1) the infusion of Ang II for 1 week increases the concentration of superoxide in aortas from rabbits. Increases in superoxide levels were observed throughout the vessel wall, but they were largest in the endothelium and adventitia. (2) The gene transfer of eNOS to the aortas of rabbits with Ang II-induced endothelial dysfunction restored responses to acetylcholine to normal. (3) The gene transfer of CuZnSOD or ECSOD did not improve relaxation to acetylcholine in aortas from rabbits with Ang II-induced endothelial dysfunction.
We chose rabbits, rather than rats, in this study because our preliminary experiments showed that the gene transfer of an adenovirus is more efficient in vessels from rabbits than rats. After the infusion of Ang II, the mean blood pressure increased to 107 mm Hg. The magnitude of the elevation of pressure was similar to that from a previous report.18 Compared with the blood pressure of rats that received Ang II (mean, 148 mm Hg),19 however, the magnitude of the blood pressure increase in rabbits was small. Nevertheless, a sustained infusion of Ang II in rabbits produced an impairment in relaxation to acetylcholine.
Lucigenin-enhanced chemiluminescence has been widely used to detect superoxide. Recent studies, however, indicated that the reduced form of lucigenin has the potential for autoxidation, with subsequent generation of superoxide.16 17 The ratio of vascular superoxide production in different groups of rabbits, however, does not depend on the lucigenin concentration that is used.17 Furthermore, we compared the ratio of vascular superoxide production using 5 μmol/L and 250 μmol/L of lucigenin. The ratio of superoxide production in aortas from Ang II–treated rabbits and saline-treated rabbits was similar (1.7) using either concentration of lucigenin. Thus, as suggested previously,17 lucigenin chemiluminescence seems to be useful for comparing relative levels of superoxide in rings from the same vessel that are exposed to different treatments.
We observed, by 2 independent methods (lucigenin and hydroethidine), an increase in the vascular levels of superoxide produced by Ang II. This finding is consistent with previous reports.8 10 Activation of an NADPH oxidase is one of the mechanisms of increasing superoxide levels by Ang II.8 This effect of Ang II may not require hypertension per se because norepinephrine, which produces similar increases in blood pressure, does not affect vascular superoxide production.8 A recent study demonstrated that NADPH oxidase is present in endothelial,20 adventitial,21 and smooth-muscle cells.7 We used hydroethidine to determine the location of superoxide after Ang II. Superoxide was increased in the endothelium and adventitia after the infusion of Ang II, as it was, albeit to a lesser extent, in smooth-muscle cells. Furthermore, the application of exogenous SOD, such as PEG-SOD, significantly decreased superoxide concentrations in the aortas from rabbits that were infused with Ang II. These findings led us to determine whether the gene transfer of SOD might reduce superoxide levels and improve vascular function.
CuZnSOD is an isoform of SOD that is present in the cytoplasm and is involved in the dismutation of superoxide within cells.22 ECSOD is the only isoform of SOD that is released from cells into the extracellular space.23 Although the concentration of ECSOD is small in most tissues, blood vessels have a large amount of ECSOD.24 Thus, ECSOD may play an important role in the regulation of vascular tone. We have reported that, after the in vitro gene transfer of β-gal to normal rabbit aortas, ≈10% of endothelial cells and 25% of adventitial cells demonstrate histochemical staining for β-galactosidase.25 Similarly, after the adenoviral-mediated gene transfer of CuZnSOD and ECSOD, overexpression of transgene is confined to the endothelium and adventitia.9
Gene transfer of CuZnSOD and ECSOD to the carotid arteries of normal rabbits significantly increases SOD activity (2.8 times and 2 times more than β-gal, respectively), and the response to acetylcholine in the carotid arteries from normal rabbits is augmented after the gene transfer of CuZnSOD.9 Neither CuZnSOD nor ECSOD, however, improved the vasomotor dysfunction in rabbits that received Ang II in this study. One possibility is that, because the gene transfer to vessels is limited to the endothelium and adventitia, SOD can dismutate superoxide in the endothelium and adventitia of the vessel wall, but not in the media. Findings with hydroethidine indicated that the media, as well as the endothelium and adventitia, of aortas from rabbits that received Ang II generated superoxide. The finding that the gene transfer of SOD does not improve responses to acetylcholine suggests that the superoxide generated by the media, which is not transduced by the gene transfer of SOD, may play a critical role in the impaired responses to acetylcholine. These findings are similar to those in atherosclerotic rabbits in which superoxide levels were increased throughout the aortic wall but in which the gene transfer of CuZnSOD or ECSOD failed to improve relaxation to acetylcholine.11 In contrast, PEG-SOD augmented the response to acetylcholine. It is likely that PEG-SOD is more effective than the gene transfer of CuZnSOD and ECSOD in improving responses to acetylcholine in aortas from Ang II–treated rabbits because PEG-SOD penetrates the entire vascular wall.
In contrast to the effects of the gene transfer of CuZnSOD and ECSOD, the gene transfer of eNOS greatly improved responses to acetylcholine in aortas from rabbits that were treated with Ang II. The augmented response is mediated by NO; L-NNA completely blocks the relaxation. Several reports indicate that the gene transfer of eNOS to normal vessels augments the response to acetylcholine.14 26 Regarding the gene transfer of eNOS, we previously confirmed the efficacy of the staining for eNOS using immunocytochemistry in carotid artery from normal rabbits that were transduced with eNOS.14 Gene transfer of eNOS to the adventitia of carotid arteries from rabbits is sufficient to augment the response to calcium ionophore.14 It is of interest that eNOS improved responses to acetylcholine, although SOD failed to improve responses. We speculate that the gene transfer of eNOS may be more effective than that of SOD because the reaction of NO with superoxide is more rapid than the response of SOD with superoxide13 and because the NO generated by the gene transfer of eNOS may diffuse more efficiently than SOD through the wall of the aorta to decrease superoxide concentrations.
Recent studies indicate that Ang II stimulates the synthesis of eNOS and induces NO production in the vascular endothelium.27 This effect of Ang II on endothelial cells may attenuate its contractile effect on smooth muscle cells.28 Attenuation of responses to acetylcholine after the infusion of Ang II in this and other studies,8 despite the increased synthesis of eNOS,27 emphasizes the effectiveness of the superoxide that is generated by Ang II in attenuating responses to acetylcholine.
Another possibility for the improvement of Ang II-induced vasomotor dysfunction by gene transfer of eNOS relates to the finding that Ang II increases the production of endothelin.29 Endothelin generally produces vasoconstriction by activating endothelin A receptors. Endothelin also stimulates the generation of NO through the activation of endothelin B receptors30 ; NO then modulates vasoconstriction by an autocrine feedback mechanism.31 Thus, we speculate that excess generation of NO by the gene transfer of eNOS may reduce Ang II-induced endothelial dysfunction, both by augmenting NO levels and attenuating the endothelin-mediated constrictor effect.
It seems that the primary mechanism by which Ang II produces endothelial dysfunction is by generating superoxide, perhaps via NADPH oxidase,8 rather than by elevating arterial pressure.10 Nevertheless, we cannot exclude the possibility that increases in arterial pressure produced by Ang II may contribute to endothelial dysfunction. For example, it is possible that the elevation of arterial pressure by Ang II may impair eNOS, and the gene transfer of eNOS may then improve endothelial function.
In conclusion, Ang II increased levels of superoxide throughout the wall of the aorta. Gene transfer of CuZnSOD and ECSOD did not improve vasomotor dysfunction in aortas from rabbits that received Ang II. Gene transfer of eNOS, however, restored the response to acetylcholine. We speculate that the NO produced by the gene transfer of eNOS may decrease superoxide in aortas and improve the vasomotor dysfunction produced by angiotensin II.
We thank Robert M. Brooks II and Jonathan Mozena for their technical assistance. We thank Dr Beverly L. Davidson and Richard D. Anderson, University of Iowa Gene Transfer Vector Core, Iowa City, for preparing the viruses. This study was supported by grants from the National Institutes of Health (HL-16066, NS-24621, HL-14388, and HL-03669), the Veterans Administration, and the American Heart Association (Grant-in-Aid).
- Received June 20, 1999.
- Revision received August 19, 1999.
- Accepted September 29, 1999.
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