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【摘要】
Objectives- Extracellular superoxide dismutase (ecSOD) lowers superoxide anions and maintains vascular nitric oxide level. We studied the function of ecSOD in high-volume hypertension induced by the 1-kidney-1-clip model in wild-type, ecSOD -/- mice, and endothelial nitric oxide synthase (eNOS) -/- mice.
Methods and Results- The 1-kidney-1-clip model resulted in impaired endothelium-dependent relaxation and hypertension and vascular oxidative stress in wild-type and ecSOD -/- mice. Recombinant ecSOD lowered the blood pressure and improved aortic nitric oxide bioavailability in wild-type and ecSOD -/- but not eNOS -/- mice. ecSOD had no effect on blood pressure in eNOS -/- or wild-type mice treated with a nitric oxide synthase inhibitor. The 1-kidney-1-clip model markedly induced ecSOD protein expression, whereas activity was increased by only 25%, suggesting a partial inactivation of ecSOD in high-volume hypertension. Incubation of aortic segments with peroxynitrite or hydrogen peroxide attenuated ecSOD activity, but peroxynitrite did not induce tyrosine nitration of ecSOD, suggesting oxidative inactivation of the enzyme. Administration of polyethyleneglycol-catalase for 3 days selectively lowered the blood pressure in ecSOD +/+ but not ecSOD -/- mice and improved nitric oxide bioavailability. In contrast, acute application of catalase had no effect.
Conclusions- Nitric oxide mediates the vascular effects of ecSOD. Vascular dysfunction in 1-kidney-1-clip model hypertension is partially a consequence of inactivation of ecSOD by reactive oxygen species.
Effects of human extracellular superoxide dismutase (ecSOD) were determined in the 1-kidney-1-clip-model (1K1C). ecSOD lowered blood pressure and improved endothelial function in ecSOD +/+ and ecSOD -/- but not eNOS -/- mice. EcSOD expression increased in 1K1C but the protein was partially inactive from the reaction with peroxynitrite and hydrogen peroxide.
【关键词】 endothelium hypertension oxidative stress superoxide dismutase
Introduction
Oxidative stress is a feature of all cardiovascular diseases and it is thought that reactive oxygen species (ROS) contribute to the initiation and progression of the disease process. See page 442
Among ROS, superoxide anions (O 2 - ), have a prominent position. Most other ROS are formed from reactions of O 2 -, and O 2 - also limits the bioavailability of nitric oxide (NO), thereby promoting endothelial dysfunction. Three superoxide dismutases (SOD) are involved in the detoxification of O 2 - to hydrogen peroxide (H 2 O 2 ). MnSOD is localized in mitochondria, CuZnSOD is expressed in the cytosol and extracellular SOD (ecSOD) is synthesized in the endoplasmic reticulum and is secreted into the interstitial space. There, the enzyme binds to matrix components such as heparin sulfate proteoglycans and fibulin-5. 1 The matrix content of most organs is relatively low and thus in most organs CuZnSOD activity is several orders of magnitudes higher than that of ecSOD. 2 Vascular structures, however, contain large amounts of matrix rendering them the tissue with the highest ecSOD activity in the body. 3 Consequently, a role of endogenous ecSOD for controlling NO bioavailability has been demonstrated in animal experiments 4 and a mutation of ecSOD which attenuates binding of the enzyme to matrix, is associated with an increased risk of cardiovascular fatalities. 5
It is unclear whether pharmacological application of human ecSOD confers protection against oxidative stress in the vasculature. Most studies reporting a beneficial effect were performed in rats. 6-8 Endogenous ecSOD in rats has a low affinity for heparan sulfate, and shows very low levels in the arterial wall. 3 This is because the enzyme is a dimer in rats, whereas it is a tetramer in other mammals. 9 Matrix binding is absolutely required for the positive effects of ecSOD, 10,11 and thus rats are almost a physiological knockout model for this enzyme. In mice with renovascular hypertension elicited by the 2-kidney-1-clip (2K1C) model, we previously demonstrated that application of ecSOD failed to improve vascular function in wild-type (WT) mice but not in knockout mice. 4 However, it has been demonstrated that in rabbit models of balloon angioplasty ecSOD application prevents inward remodeling, 12 suggesting that dependent on the model, exogenously applied ecSOD might be beneficial.
The effects in the 2K1C model are mediated by the renal release of renin, which leads to the generation of angiotensin II. We have previously demonstrated that the endothelial dysfunction in 2K1C renovascular hypertension in mice is exclusively mediated by the endothelial production of O 2 -. 13 In the balloon injury model, O 2 - production is accompanied by a concomitant increase in NO production, resulting in oxidative as well as nitrosative stress and the formation of peroxynitrite. 12 We therefore hypothesize that NO has a central role in determining the effect of SOD application. To test this hypothesis, we studied the vascular function in the 1-kidney-1-clip (1K1C) model of renovascular hypertension in WT, ecSOD -/-, and endothelial nitric oxide synthase (eNOS) -/- mice. The 1-kidney-1-clip model induces nonrenin, low-angiotensin II hypertension, which is mediated by salt and water retention and thus resembles a situation of high-volume hypertension with increased cardiac output and NO formation. 14
Materials and Methods
Animals and 1-Kidney-1-Clip Model
C57/b6-ecSOD -/- and C57/b6-ecSOD +/+ mice were obtained from the breeding facility in Umea, Sweden, and C57/b6-eNOS -/- mice were kindly provided by Axel Gödecke, Heinrich-Heine-Universität Düsseldorf. The animals were bred at the local animal facility at Frankfurt Medical School. Only male mice were used for this study. At an age of 8 weeks, animals were subjected to sham operation or renal artery clip application of the left kidney as described previously. 4,14 A sham procedure, which included the entire surgery with the exception of artery clipping, was performed in control mice. 14 On the third day after operation, the right kidney was removed using a similar operation technique in the sham as well as the clipped group. Lethality of the procedure was 30% in the first 5 days after the operation in the clipped group, whereas subsequently no further animals died. Blood pressure was measured 14 and 28 days after the operation and organ chamber experiments were performed 29 days after the operation. Successful 120 mm Hg 2 weeks after clip application was observed in 70% of the animals. Animals that did not meet this criterion were excluded from the study. Experiments conformed to the guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH publication 85- 23) and were approved by the local government (II25.3-19c20/15-F61/16).
Blood Pressure Measurements and ecSOD Application
Mice were anesthetized using isoforene and catheters were place in the right common carotid artery and the left external jugular vein. Blood pressure was measured continuously using a Statham pressure transducer. Human recombinant ecSOD (100 000 U/kg, provided by S.L. Marklund), PEG-catalase (Sigma, Deisenhofen, Germany; 100 000 U/kg) and N -nitro- L -arginine methyl ester (30 mg/kg) were applied as a bolus (dissolved in 100 µL of normal saline) via the vein catheter as described previously. 4 In subgroups PEG-catalase was administered for 3 days by daily injection of the aforementioned dose into the tail vein. Oxonic acid dissolved in water was delivered by Alez minipumps (obtained via Charles Rivers, model 2001) inserted into a subcutaneous pouch between the scapulae for seven days at a rate of 2.8 mg/kg per day.
Organ Chamber Experiments
Organ chamber experiments were performed as described. 15 Relaxations to cumulatively increasing concentrations of acetylcholine or deta-NONOate were recorded in vessels preconstricted to 80% of the maximal KCl (80 mmol/L)-induced contraction using phenylephrine in the presence of diclofenac (10 µmol/L). Relaxations are denoted as percent of the initial constriction obtained by phenylephrine. NO bioavailability was estimated from the constrictor response to the NO synthase inhibitor N -nitro- L -arginine (300 µmol/L) in aortic rings preconstricted to 10% of the maximal KCl constriction using phenylephrine.
Vascular Radical Generation
Measurements were performed using a lucigenin (5 µmol/L)-enhanced chemiluminescence assay in intact mouse aortic rings as described previously. 15
SOD Activity
For activity determination, 3 whole mouse aorta were pooled per data point and total SOD activity, ConA-sepharose-binding SOD activity (ecSOD), and cyanide-sensitive SOD activity (CuZn-SOD) were determined by the potassium superoxide assay as described previously. 3,16
Immunoblotting
Western blot analysis from Triton x-100 (1%) soluble aortic protein was performed as described previously. 15 The rabbit anti-MnSOD antibody was from Dr W. Gwinner (Medizinische Hochschule Hannover, Germany). The following commercially available antibodies were used: mouse anti-eNOS (BD Transduction), sheep anti-CuZnSOD (Calbiochem), and rabbit anti-catalase (Calbiochem).
Statistics
All values are mean±SEM. Maximal relaxations were calculated from individual dose-response curves. Statistical analysis was performed using 1-way analysis of variance for repeated measurements followed by Fisher least significant difference test, or paired t test, if appropriate. Values of P <0.05 were considered statistically significant.
Results
Vascular Dysfunction in 1K1C Hypertension Is Independent of Endogenous ecSOD
The 1K1C model resulted in a marked increase in systolic blood pressure as determined by tail-cuff technique in WT mice ( Figure 1 A). Moreover, endothelium-dependent relaxation was blunted in isolated aortic rings obtained from 1K1C hypertensive WT mice as compared with segments from control animals ( Figure 1 B), and this was 3-fold increase in vascular O 2 - concentrations as determined by lucigenin chemiluminescence ( Figure 1 C). This was accompanied by formation of peroxynitrite as aortic protein tyrosine nitration was increased in 1K1C hypertensive mice (data not shown).
Figure 1. Effect of the 1-kidney-1-clip model in ecSOD +/+ and ecSOD -/- mice. Male ecSOD +/+ and ecSOD -/- were subjected to right-sided nephrectomie and left-sided renal artery clip application (clip) or sham procedure (sham). After 4 weeks, the systolic blood pressure was measured by tail cuff technique (A). Endothelium-dependent relaxation to acetylcholine (ACh) (B) and superoxide anion level by lucigenin chemiluminescence (C) were determined in aortic segments. * P <0.05, n=8, BP indicates blood pressure; CPM, counts per minute.
O 2 - concentrations in aortic segments from sham-operated ecSOD -/- mice were significantly higher even than those determined in segments from 1K1C-operated ecSOD +/+ mice. In comparison to aortic rings from sham-operated ecSOD -/- mice, O 2 - concentrations in segments from 1K1C were further enhanced ( Figure 1 C). Interestingly, despite this difference in O 2 - concentrations endogenous ecSOD appears to be less important for hypertension development and endothelial dysfunction in the 1K1C model. No difference in the hypertensive response to the clip application was observed between ecSOD +/+ and ecSOD -/- mice and the extent of the procedure-induced attenuation of endothelium-dependent relaxation was identical in both strains. However, acetylcholine-induced relaxation in aortic segments from sham-operated ecSOD -/- mice was slightly smaller than that observed in vessels from ecSOD +/+ mice and this difference was maintained in the 1K1C group despite a marked attenuation of the endothelium-dependent relaxation ( Figure 1 B).
These observations are in striking contrast to the data previously obtained by our group in the 2K1C model of renovascular hypertension. In this model blood pressure was significantly higher in clipped ecSOD -/- than ecSOD +/+ mice and aortic endothelial dysfunction was more pronounced in clip-operated ecSOD -/- than ecSOD +/+ mice. 4
Interestingly, as compared with the level previously measured in ecSOD +/+ mice subjected to the 2K1C protocol, the 1K1C procedure resulted in markedly higher vascular O 2 - concentrations in the same strain (ecSOD +/+ : sham, 39±12; 2K1C: 79±24 cmp; 1K1C, 167±9 cmp; P <0.05, 2K1C versus 1K1C, n=8). This difference, however, was not observed in the absence of ecSOD, because both models increased the radical production to the same level in ecSOD -/- mice (ecSOD -/- : sham, 249±66; 2K1C, 442±78; 1K1C, 404±126 cpm; P =not significant, 2K1C versus 1K1C, n=8) ( Figure 1 C).
Human Recombinant ecSOD Improves Vascular Function in 1K1C Hypertension
Because no difference between ecSOD +/+ and ecSOD -/- mice was observed after the 1K1C procedure, it was tested whether in this form of hypertension ecSOD plays any role for vascular function. When human recombinant ecSOD was administered intravenously the mean blood pressure recorded in the carotid artery declined 15 mm Hg. The blood pressure lowering effect of ecSOD, however, was identical between ecSOD +/+ and ecSOD -/- mice ( Figure 2 A). Similar observations were made in isolated aortic segments: In vivo application of human recombinant ecSOD improved acetylcholine-induced relaxation as well as basal NO formation in aortic segments from animals subjected to the 1K1C procedure. The effect, however, was identical between ecSOD +/+ and ecSOD -/- mice ( Figure 2B, 2 C). These observations are in striking contrast to the data we previously obtained in the 2K1C model, in which only ecSOD -/- but not ecSOD +/+ mice responded to ecSOD application. One possible explanation for this finding could be that the endogenous ecSOD is inactivated or downregulated during 1K1C hypertension.
Figure 2. Effect of human recombinant ecSOD on mice with 1-kidney-1-clip hypertension. (A) Mean blood pressure as determined by carotid artery catheter before and after intravenous application of 100 000 U/kg ecSOD in mice 4 weeks after 1K1C operation (n=8). Acetylcholine (ACh)-induced relaxation (B) and nitro- L -arginine (L-NA)-induced contraction (C) in aortic rings from mice 4 weeks after 1K1C with (+) and without (-) in vivo application of ecSOD 16 hours before euthanization. n=8, * P <0.05
ecSOD Is Partially Inactivated During 1K1C Hypertension
Expression and activity of the 3 isoforms of SOD were determined in aortas. 1K1C hypertension led to a massive induction of ecSOD protein expression; MnSOD protein was slightly increased, whereas almost no effect was detected for CuZnSOD ( Figure 3 A). Interestingly, these effects were not mirrored by the aortic ecSOD activity, which increased by 25% ( Figure 3 B). These observations indicate that large amounts of the ecSOD produced in the 1K1C model are inactive.
Figure 3. Effect of the 1-kidney-1-clip model on aortic superoxide dismutase expression and activity. Protein expression as determined by Western blot analysis in ecSOD +/+ and ecSOD -/- mice (A) and activity of ecSOD +/+ mice as determined by potassium superoxide assay (B) of ecSOD, CuZnSOD and MnSOD in aortic segments from mice 4 weeks after 1K1C operation (clip) or sham procedure. ß-actin served as loading control (n=5).
Human Recombinant ecSOD Is Without Effect in eNOS -/- Mice
In a next step we determined whether the eNOS mediates the responsiveness to ecSOD in 1K1C model. These experiments turned out to be problematic as the 1K1C model resulted in an excessive mortality in eNOS -/- 80% within the first 10 days because of uremia from the loss of the clipped kidney. In surviving mice, the 1K1C model elicited an increase in blood pressure comparable to that observed in WT mice. Importantly, application of human ecSOD had no effect on blood pressure in eNOS -/- mice, suggesting that either the hypotensive effect of ecSOD requires endothelial NO or that the presence of eNOS alters endogenous ecSOD activity to render the mice sensitive to ecSOD application (Figure I, please see http://atvb.ahajournals.org).
In organ chamber experiments, the relaxations to deta-NONOate of aortic rings from eNOS -/- mice were found to be unaffected by in vivo ecSOD treatment. In contrast, relaxations to deta-NONOate in rings from ecSOD -/- and ecSOD +/+ mice were improved by in vivo ecSOD application. As the effect of ecSOD on the deta-NONO-induced relaxation involves scavenging of O 2 -, it could to be concluded that O 2 - levels are lower in aortas of eNOS -/- mice compared with control mice. A possible explanation could be that eNOS uncoupling largely contributes to the oxidative stress in the 1K1C model. Alternatively, the concomitant generation of O 2 - and NO will yield peroxynitrite (ONOO - ), which may inactivate ecSOD in WT animals. Interestingly, ecSOD expression was also much higher in eNOS -/- mice than in WT mice (Figure I), which could be another explanation for the failure of exogenous ecSOD to affect relaxations.
Despite the lack of effect of ecSOD in eNOS -/- mice, the aortic responses to deta-NONOate were largely blunted in hypertensive animals of this strain (Figure IB). It is possible that this difference is a consequence of a dysfunction of the smooth muscle cells in hypertensive eNOS -/- mice. Indeed, in preliminary experiments lower aortic expression of soluble guanylyl cyclase was observed in eNOS -/- mice subjected to the 1K1C procedure as compared with sham operated animals (Brandes RP, unpublished observation, 2006).
Hydrogen Peroxide and Peroxynitrite Inactivate ecSOD
To determine whether ecSOD is inactivated by ROS, isolated aortic segments were exposed to peroxynitrite (ONOO - ) and hydrogen peroxide (H 2 O 2 ). Both ROS decreased ecSOD activity, although the effect of ONOO - was more pronounced than that of H 2 O 2. In contrast, CuZnSOD activity was similarly impaired by ONOO - and H 2 O 2, whereas MnSOD activity remained unaffected ( Figure 4 A).
Figure 4. Effect of peroxynitrite and hydrogen peroxide on aortic SOD activity. A, Aortic segments were incubated in Hanks buffer (1 mL) in the absence (CTL) or presence of peroxynitrite (PON) (1 mmol/L stock concentration, 10 µL was applied during stirring 3 times) or H 2 O 2 (1 mmol/L, 4 hours.). Subsequently, activity of SOD isoforms was determined by the potassium superoxide assay. Three whole aortas were pooled for each data point (n), n=3 each group. B, Analysis of tyrosine nitration of ecSOD in response to peroxynitrite: Aortic segments were incubated in Hanks buffer (1 mL) for 30 minutes at 37°C. Peroxynitrite (ONOO -, 1 mmol/L stock concentration, 10 µL) or solvent (CTL) was applied during stirring 3 times. Vessels were mortared, lysed, and subjected to immunoprecipitation. PC indicates samples before immunoprecipitation; IP, immunoprecipitates; PIP, post immunoprecipitation samples; IP, antibody used for immuno-precipitation (either anti-NO-Tyr or anti-ecSOD); IB, antibody used for immunodetection on the Western blot. The blots shown are representative for 3 experiments.
To determine whether the inhibitory effect of ONOO - results from tyrosine nitration of ecSOD, immunoprecipitations was performed. These experiments, however, revealed that several proteins but not ecSOD are tyrosine-nitrated on exposure to ONOO - ( Figure 4 B), suggesting that rather thiol oxidation or the formation of amino acid radicals mediate inactivation of ecSOD.
Prolonged Treatment With Catalase Selectively Improves Vascular Function in ecSOD +/+ Mice
To determine whether H 2 O 2 contributes to the inactivation of ecSOD in vivo, mice were treated with daily injections of PEG-catalase for 3 days and studied subsequently. This prolonged treatment selectively lowered the blood pressure in ecSOD +/+ mice but was without effect in ecSOD -/- mice. Importantly, injection of ecSOD in PEG-catalase-pretreated mice only lowered the blood pressure in animals of the ecSOD -/- strain but was without effect in ecSOD +/+ mice ( Figure 5 A). In line with these observations, the endothelium-dependent relaxation of aortic rings from hypertensive ecSOD +/+ pretreated with PEG-catalase was significantly greater than that obtained in untreated hypertensive controls ( Figure 5 B).
Figure 5. Effects of in vivo treatment with PEG-catalase, L-NAME, and oxonic acid in 1K1C hypertension. A, Mean blood pressure as determined by carotid artery catheter before and after intravenous application of 100 000 U/kg ecSOD in ecSOD +/+ and ecSOD -/- mice 4 weeks after 1K1C operation. Subgroups were treated with daily injections of PEG-catalase (catalase, 40 000 U/kg) for 3 days before the catheterization (n=6). * P <0.05 before vs after ecSOD, # P <0.05 with vs without PEG-catalase. Acetylcholine (ACh)-induced relaxation in aortic rings from ecSOD +/+ mice 4 weeks after 1K1C operation without (control) and with in vivo pretreatment with PEG-catalase (B) or oxonic acid (C) (applied by osmotic minipump at a rate of 2.8 mg/kg per day over 7 days; n=8). D, Mean blood pressure before and 15 minutes after acute intravenous application of PEG-catalase (40 000 U/kg, n=4) or N -nitro- L -arginine methyl ester (L-NAME) (30 mg/kg, n=3). Subsequently, ecSOD (100 000 U/kg) were injected and the blood pressure was measured again after 15 minutes. * P <0.05 vs "before."
Hydroxyl Radical Scavenging Improves Endothelial-Dependent Relaxation in 1K1C Hypertension
It has been suggested that H 2 O 2 through the peroxidase activity of SODs leads to the generation of hydroxyl radicals, which subsequently inactivate SOD. Therefore, the plasma concentrations of the hydroxyl radical scavenger uric acid were increased by treating mice with oxonic acid for 7 days, as reported previously by others. 17 Indeed, similar to PEG-catalase, in vivo treatment of 1K1C hypertensive ecSOD +/+ mice with oxonic acid increased the endothelium-dependent relaxation of the isolated aorta ( Figure 5 C).
NO and Not Hydrogen Peroxide Mediates the Acute Blood Pressure-Lowering Effect of ecSOD
It is unclear whether the blood pressure-lowering effect of ecSOD is mediated by an increased generation of H 2 O 2 or an impaired scavenging of NO. Therefore, the effects of acute application of PEG-catalase and the NO synthase inhibitor L-NAME were compared. PEG-catalase had no effect on blood pressure, whereas L-NAME resulted in a marked increase in pressure, which was fatal for the mice within 60 minutes. ecSOD had no effect on blood pressure in L-NAME-treated mice. In contrast, in PEG-catalase-pretreated animals, the blood pressure-lowering effect was similar to that obtained in untreated 1K1C hypertensive animals ( Figure 5 D). These data clearly demonstrate that NO, but not H 2 O 2, mediates the vascular effects of ecSOD.
Discussion
In this study we observed that application of human recombinant ecSOD improves endothelial function and lowers blood pressure in a mouse model of high-volume hypertension induced by the 1K1C procedure. Therefore, direct evidence is provided that in this type of hypertension endogenous ecSOD does not provide sufficient activity to scavenge extracellular superoxide. The effects of ecSOD application were dependent on eNOS and evidence is presented that endogenous ecSOD is partially inactivated by hydroxyl radicals formed from H 2 O 2.
In arteries, ecSOD is present in large abundance 3 and is involved in the maintenance of vascular function in high-renin hypertension. Indeed, we previously observed that endogenous ecSOD attenuates the hypertensive response and endothelial dysfunction in ecSOD -/- mice with 2K1C-induced hypertension, whereas application of recombinant ecSOD failed to elicit any effects in WT mice. 4 These former observations are in line with several studies performed in rats, 6-8 which lack significant vascular ecSOD activity. 11 In contrast to this, it was observed that in rabbits, a species with high-endogenous ecSOD activity, application of ecSOD either as recombinant enzyme 12 or by gene therapy 18,19 can have positive effects. This difference suggests that the pathophysiological situation determines whether ecSOD application elicits positive vascular effects and whether endogenous ecSOD is sufficient to dismute the majority of extracellular O 2 -. Indeed, different to the 2K1C model, the studies performed in rabbits utilized disease models with concomitant formation of O 2 - and NO. Therefore we hypothesized that the interplay of these two radicals determines the sensitivity to exogenous NO. Consequently, the role of ecSOD for vascular function in a mouse model of oxidative and nitrosative stress, the 1K1C model of hypertension was studied.
In the 1K1C model, hypertension is maintained by a nonrenin-dependent mechanism after the second week after surgery, 14,20 which is very much different to the 2K1C model of hypertension. In the latter model, the obstruction of one renal artery increases the renin release and the subsequent formation of angiotensin II and aldosterone increases the blood pressure. As hypertension induces pressure diuresis through the nonclipped kidney, the model remains renin-dependent for a long period. In the 1K1C model, however, the nonclipped kidney is removed and thus hypertension-induced diuresis is prevented leading to retention of sodium and water. The more volume and cardiac output increases in the 1K1C model, the less it becomes dependent on renin till eventually normal perfusion of the kidney is restored at normal renin but high volume level. 14 At that stage, which is usually reached after 2 weeks, blockade of the AT1 receptor does not affect blood pressure in rats 21 or 8-isoprostane level. 22 The effects of high-volume hypertension on the vascular radical formation have not been extensively studied, and despite the observation that antioxidants lower the blood pressure in the 1K1C model, the enzymatic sources of ROS are currently unknown. 22 Although the 1K1C model has similarities to the DOCA and salt model of high-volume hypertension, sodium retention is achieved via a mineralocorticoid-independent mechanism in the 1K1C model. This aspect might be important as mineralocorticoids activate the NADPH oxidase 23 and as the mineralocorticoid antagonist spironolactone lowers vascular superoxide anion formation in some disease models. 24 Both high-volume models are however characterized by an activation of the endothelin system and endothelin-1 via activation and/or induction of the NADPH oxidase seems to contribute to oxidative stress. 25-27 Moreover, uncoupling of eNOS by peroxynitrite appears to be a mechanism to increase vascular O 2 - production and to decrease NO formation in high-volume hypertension. 28 Accordingly, application of sepiapterin slightly but significantly improves endothelium-dependent relaxation in aortic rings from mice subjected to the 1K1C model (Jung et al, unpublished observation 2006). Besides endothelin, the trigger for vascular O 2 - production in the 2 models is unknown. It appears however possible that hypertension per se increases oxidative stress as indicated in studies using the aortic banding model. In these studies, arteries distal of the stenosis had low vascular O 2 - production, whereas O 2 - production was increased in the vascular areas exposed to hypertension and this is associated with the induction of NADPH oxidase subunits and protein kinase C. 29-31 Also endothelial NO formation is increased in response to enhanced circumferential tension, 32 and peroxynitrite formation has uniformly been observed in models of high-volume hypertension. 22,28
The data obtained with eNOS -/- mice in this study suggest a critical role of endothelial NO for the action of recombinant ecSOD. eNOS-derived ONOO - could potentially inactivate abundantly expressed endogenous ecSOD, rendering the system sensitive to the exogenously applied enzyme. eNOS could also be a major source of O 2 - in the present study and finally, the vasodilator effects of ecSOD could be mediated by increasing basal NO systemically, leading to direct vasodilatation. Our observations suggest that all these mechanisms are functional in high-volume hypertension. Acute inhibition of NOS completely blocks the effects of recombinant ecSOD on blood pressure, proving that the effect on blood pressure is mediated by increasing the NO bioavailability rather than that of H 2 O 2. However, in 1K1C hypertensive eNOS -/- mice, in vivo treatment with recombinant ecSOD had no effect on the deta-NONOate-induced relaxation of isolated aortic segments, whereas the same approach improved dilator responses in ecSOD +/+ as well as ecSOD -/- mice. This observation is indicative toward an inactivation of endogenous ecSOD by a mechanism involving eNOS. Indeed, evidence has been presented to suggest that ecSOD inactivation occurs under certain disease conditions. In a rabbit balloon injury model, a significant decrease in ecSOD activity was observed despite an increase in protein levels. 12 Moreover, in apoE -/- mice, inactivation of ecSOD was detected. Increasing the plasma concentration of urate, a scavenger of hydroxyl radicals and ONOO -, restored vascular ecSOD activities. 17 The physiological relevance of ecSOD inactivation and the effect of different ROS on aortic ecSOD activity were not determined so far. The present observation that prolonged in vivo treatment with catalase selectively lowers the blood pressure in hypertensive ecSOD +/+ mice and improves ex vivo aortic endothelial function clearly demonstrate that H 2 O 2 is central for the inactivation of endogenous ecSOD. The effects of H 2 O 2 appear to be mediated by hydroxyl radicals: When hypertensive mice were treated with oxonic acid for 7 days, the ex vivo aortic endothelial vasomotor function improved. In pilot experiments we also observed that oxonic acid lowered the blood pressure in hypertensive mice (Xia, unpublished observations, 2006). Oxonic acid inhibits uricase, and thus increases urate level in mice, 17,33 and urate is known to scavenge hydroxyl radicals as well as ONOO -. 34 Despite the lack of selectivity of uric acid, data to suggest an involvement of ONOO - for the present disease mechanisms could not be obtained. The mechanism of H 2 O 2 -mediated inactivation of Cu-containing SODs is relatively well understood and involves the peroxidase activity of the enzyme itself. 17 ONOO -, in contrast could act via tyrosine nitration or oxidation of proteins. In the present study we found no evidence for tyrosine nitration of ecSOD using immunoprecipitation. It has been demonstrated that the inactivation of CuZnSOD by ONOO - involves histidyl radicals rather than tyrosine nitration. 35 In addition to this aspect it is important to realize that under physiological conditions, ONOO - will preferentially react with abundantly expressed antioxidants such as glutathione or will mediate its effect through the formation of secondary radicals. 36 Consequently, based on the present data it is impossible to role out but also to prove some contribution of ONOO - in the present model.
Blood pressure in the present study was determined using 2 different methods: tail cuff technique in conscious animals and carotid artery catheter in anesthetized mice. Using both approaches it was observed that the blood pressure between ecSOD -/- and ecSOD +/+ mice was identical. Both techniques, however, have substantial limitations and thus it cannot be excluded that slight differences that could only be detected by telemetry in large groups were overseen. This aspect does not relate to the central finding of this study that ecSOD improves vascular function, because this effect was also observed in isolated vessels and therefore was independent of any confounders in the blood pressure methods.
Interestingly, aortic ecSOD expression increased in the 1K1C model. It has been demonstrated previously that endothelial NO 37 as well as angiotensin II via a pathway not involving oxidative stress 38 increase ecSOD expression. It is unlikely that these 2 mechanisms contribute to the induction observed in the present study, because angiotensin II level are low in 1K1C hypertension (Jung O, 2006, unpublished observation) and because NO bioavailability was low through O 2 - -mediated scavenging. Moreover, particular in eNOS -/- mice, aortic ecSOD expression was high and further increased by the clip procedure. These data are in contrast to a previous observation 37; still, the 1K1C-mediated increase in ecSOD expression in eNOS -/- mice excludes endothelial NO to contribute to the induction observed in the present study. In cultured smooth muscle cells, NO rather decreases than increases ecSOD expression. 39 It has recently been reported that the increased expression of ecSOD observed in apoE -/- mice is attributable to high expression of Atox-1. 40 Therefore, it is attractive to speculate that a similar mechanism might be function in eNOS -/- mice as well as in 1K1C hypertension.
In conclusion, the present study provides evidence that shortage of endogenous vascular ecSOD activity occurs in situations of combined oxidative and nitrosative stress, and that replenishment of the vascular ecSOD activity pool by application of ecSOD or SOD mimetic might lead to normalization of endothelial function in hypertensive patients.
Acknowledgments
The authors thank Tanja Schönfelder, Sina Bätz, and Katalin Palfi for outstanding expert technical assistance. We are grateful for the excellent service provided by the team of the animal care facility.
Sources of Funding
This study was supported by grants from the Deutsche Forschungsgemeinschaft to R.P.B. (BR1839/2-3 and BR1839/3-1) and the Herbert and Hedwig Eckelmann-Foundation to O.J.
Disclosures
None.
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作者单位：From Institut für Kardiovaskuläre Physiologie (O.J., N.X., R.B., R.P.B.) and Medizinische Klinik IV (O.J.), Funktionsbereich Nephrology, Klinikum der J.W.-Goethe-Universität, Frankfurt am Main, Germany; and the Department of Medical Biosciences (S.L.M.), Clinical Chemistry, Umea Unive