This Article Abstract Full Text (PDF) Data Supplement Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, L. Articles by Chen, A. F. Search for Related Content PubMed PubMed Citation Articles by Li, L. Articles by Chen, A. F. Related Collections Hypertension - basic studies Gene therapy Oxidant stress Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:249.)
© 2002 American Heart Association, Inc.

Vascular Biology

Gene Transfer of Endothelial NO Synthase and Manganese Superoxide Dismutase on Arterial Vascular Cell Adhesion Molecule-1 Expression and Superoxide Production in Deoxycorticosterone Acetate-Salt Hypertension

Lixin Li; Elahe Crockett; Donna H. Wang; James J. Galligan; Gregory D. Fink; Alex F. Chen

From the Departments of Pharmacology and Toxicology (L.L., J.J.G., G.D.F., A.F.C.), Surgery (E.C.), and Medicine (D.H.W.) and the Neuroscience Program (L.L., J.J.G., G.D.F., A.F.C.), Michigan State University, East Lansing.

Correspondence to Dr Alex F. Chen, Assistant Professor, Department of Pharmacology and Toxicology and the Neuroscience Program, College of Human Medicine, Michigan State University, East Lansing, MI 48824-1317. E-mail chenal{at}msu.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Enhanced vascular cell adhesion molecule-1 (VCAM-1) expression directly contributes to vascular dysfunction in hypertension. Decreased NO and/or increased superoxide are causative factors for such an event in the vessel wall. The present study was undertaken to determine whether gene transfer of endothelial NO synthase (eNOS) or manganese superoxide dismutase (MnSOD) affects VCAM-1 levels in arteries from hypertensive rats. Isolated carotid and femoral arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats were transduced for 4 hours with adenoviral vectors encoding eNOS, MnSOD, or ß-galactosidase reporter genes. Recombinant eNOS or MnSOD expression was evident morphologically and quantitatively 24 hours after gene transfer. Immunohistochemistry, ELISA, and Western blot techniques were used to determine VCAM-1 expression and levels. In addition, endogenous eNOS and MnSOD and in situ superoxide levels were analyzed by immunoblotting and fluorescence confocal microscopy, respectively. Arterial VCAM-1 expression was significantly higher in DOCA-salt hypertensive rats than in sham-operated rats; this expression was accompanied by decreased MnSOD but unaltered endogenous eNOS levels. VCAM-1 expression was significantly lower in MnSOD- and eNOS-transduced hypertensive arteries, with a concomitant reduction of superoxide level. These results suggest that gene transfer of MnSOD or eNOS suppresses arterial VCAM-1 expression in DOCA-salt hypertension by reducing the superoxide level.

Key Words: endothelial NO synthase • gene transfer • hypertension • superoxide • vascular cell adhesion molecule-1

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Although hypertension is one of the key risk factors for atherosclerosis, the underlying molecular and cellular mechanisms remain to be delineated.¹ Enhanced adhesion molecule expression is known to contribute directly to vascular dysfunction in hypertension.² Vascular cell adhesion molecule-1 (VCAM-1) is an early marker of endothelial activation and dysfunction, leukocyte infiltration, and vascular remodeling in the development of early atherosclerotic lesions (fatty streaks and fibrous plaques).^2–5 Although VCAM-1 is structurally similar to intercellular adhesion molecule-1 and other adhesion molecules, its pattern of expression is unique. It exhibits low to negligible expression under baseline conditions but is profoundly upregulated by proatherosclerotic conditions in animal models and in humans.^2–5 In addition, compared with other adhesion molecules whose expression often extends into uninvolved and/or lesion-protected regions of the vessel wall, VCAM-1 expression is largely restricted to atherosclerotic lesions and lesion-predisposed regions.^2–5 Consistent with these phenomena, a recent study has demonstrated that VCAM-1, but not intercellular adhesion molecule-1 (ICAM-1), plays a critical role in early atherogenesis.⁶

Enhanced adhesion molecule expression has been ascribed to an imbalance between oxidative stress and antioxidant activity. Experimental evidence suggests that NO and superoxide are 2 key regulating factors for the expression of adhesion molecules, including VCAM-1.^7,8 In angiotensin II (Ang II)–induced hypertension, Ang-II stimulates vascular NADPH oxidase to produce superoxide, which not only inactivates NO and impairs vasomotor function^9,10 but also contributes to atherogenesis by the activation of VCAM-1.¹¹ The elevated superoxide is present throughout the atherosclerotic vessel wall,¹² and a major source of superoxide is from the electron transport chain in mitochondrial respiration.¹³

Two recent studies have indicated that there is also a substantial increase of vascular superoxide with reduced NO release in deoxycorticosterone acetate (DOCA)-salt hypertension,^14,15 a model well known for its suppressed plasma renin and angiotensin levels.¹⁶ However, the level of vascular VCAM-1 expression has never been investigated in this model of hypertension.

Therefore, the present study was undertaken to address the following 3 unanswered questions: (1) Is there an increase of VCAM-1 in carotid and femoral arteries of DOCA-salt hypertensive rats? (2) Are there altered levels of endogenous endothelial NO synthase (eNOS) and manganese superoxide dismutase (MnSOD), the mitochondrial scavenging isozyme? (3) What are the effects of ex vivo gene transfer of eNOS and MnSOD on VCAM-1 and superoxide in DOCA-salt hypertension? Our results showed that VCAM-1 and superoxide levels were significantly increased in carotid and femoral arteries, with a concomitant decrease in the endogenous MnSOD level; these responses were ameliorated after MnSOD or eNOS gene transfer.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
DOCA-Salt Hypertension
DOCA-salt hypertension was created in male Sprague-Dawley rats as previously described.¹⁷ Briefly, rats (250 to 275 g, Charles River Laboratories, Portage, Ind) underwent uninephrectomy (flank incision, left side), and a silicone rubber DOCA implant (200 mg/kg) was placed subcutaneously between the shoulder blades. Sham-operated rats were also uninephrectomized but received no implant. DOCA-salt rats received 1.0% NaCl and 0.2% KCl in water to drink, and sham-operated rats received tap water. All animals were fed standard rat chow and had ad libitum access to food and drinking solution. Hypertension develops gradually in this model, with arterial pressure rising gradually but steadily over a 4-week period.¹⁷ Blood pressure was measured by using noninvasive tail-cuff measurements in conscious but restrained and prewarmed rats. All the vessels used were collected between weeks 4 and 6 after DOCA implantation. All animal procedures were in accordance with the institutional guidelines of Michigan State University.

Preparation of Adenoviral Vectors
The propagation, purification, and titration of replication-incompetent adenoviral vectors were routinely performed as previously described.^18–20 The prepared ß-galactosidase (ß-Gal), eNOS, and MnSOD vectors were stored at -80°C in 0.01 mol/L Tris, 0.01 mol/L MgCl₂, and 10% glycerol before use.

Ex Vivo Gene Transfer
Isolated arterial segments (4 mm) were transduced with adenoviral vectors with titers between 10¹⁰ to 10¹¹ plaque-forming units (pfu)/mL as indicated in MEM at 37°C for 4 hours, followed by incubation in fresh medium for 24 hours as previously described.^18,19 Arteries transduced with the ß-Gal marker gene served as controls.

Immunohistochemistry for VCAM-1 and Recombinant Protein Expression
Immunohistochemistry was performed as described.^18,19 Briefly, cross sections of the vessel (6 µm thin) were fixed in ice-cold acetone for 10 minutes, and endogenous peroxidase was inhibited with 0.3% (vol/vol) hydrogen peroxide for 30 minutes. Sections were blocked with 5% horse serum/PBS-Tween 20 (pH 7.4) for 20 minutes and incubated with primary antibodies at room temperature for 2 hours, each diluted in PBS-Tween 20 containing 2% horse serum. The primary antibodies used were goat polyclonal antibody for VCAM-1 (1:40, Santa Cruz Biotechnology), mouse monoclonal antibody for eNOS (1:125, Transduction Laboratories), and sheep monoclonal antibody for MnSOD (1:200, Biodesign International). Nonimmune goat IgG₁ was used as a negative control at the same concentrations that were used for the primary antibodies. Sections were then incubated with biotinylated secondary antibodies diluted at 1:750 in PBS-Tween 20 containing 2% horse serum (Vector Laboratories). Visualization was performed with an AEC kit (Vector Laboratories). Nuclei were counterstained with hematoxylin.

ELISA for VCAM-1
Arteries were isolated and immediately stored in liquid nitrogen. The frozen segments were pulverized and solubilized in lysis buffer (100 mmol/L K₂HPO₄, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.2% Triton X-100). Arteries were homogenized on ice. This procedure was followed by centrifugation at 14 000g for 20 minutes to remove the insoluble pellet, and protein concentration was determined by Bio-Rad DC protein assay. ELISA for VCAM-1 was performed as described.²¹ Dilutions of supernatant were incubated overnight in microplates. Dried supernatants were incubated with PBS/1% BSA/0.02 azide at 37°C for 1 hour, washed once in PBS with 0.02% azide, incubated with the VCAM-1 antibody (1:20) at 37°C for 1 hour, and finally washed 3 times in PBS containing 0.05% Tween-20. Alkaline phosphatase–conjugated secondary antibody (1:10 000, Sigma Chemical Co) was added at 37°C for 1 hour and then washed out. Dilute substrate in diethanolamine buffer (Sigma) was added for 15 minutes, and the reaction was stopped by the addition of 0.1 mol/L EDTA. Absorbance at 405 nm was read with the use of an EL340 microtiter plate reader (Bio-Tek⁺ Instruments).

Western Immunoblot for VCAM-1, eNOS, and MnSOD
The detailed procedures have been described previously.²⁰ Arteries were cleaned and homogenized in lysis buffer (100 mmol/L K₂HPO₄, 1 mmol/L phenylmethylsulfonyl fluoride, and 0.2% Triton X-100). Equal amounts of protein preparations (10 µg in 25 µL buffer) were run on SDS-polyacrylamide (7.5% for VCAM-1 and eNOS, 12% for MnSOD) gels, electrotransferred to polyvinylidine difluoride membranes, and blotted with a primary antibody against VCAM-1 (1:500), eNOS (1:1500), and MnSOD (1:150). The secondary antibodies used were bovine anti-goat antisera for VCAM-1 and MnSOD (1:5000) and rabbit anti-mouse for eNOS (1:4000). Blots were developed by using ECL plus reagent (Amersham Pharmacia Biotech). Prestained protein marker was used for molecular mass determinations. To confirm equal protein loading of all samples, membranes were stained with Coomassie brilliant blue R-250 (Sigma). Molecular band intensity was determined by densitometry (NIH image software).

In Situ Detection of Superoxide
In situ detection of superoxide anion was performed by confocal microscopy with the use of the oxidative fluorescent dye dihydroethidium, as described previously.¹² Dihydroethidium is freely permeable to cell membranes and fluoresces red when oxidized to ethidium bromide (EtBr) by superoxide. Unfixed frozen vessel sections (30 µm) with or without gene transfer were placed on glass slides and submerged in 10^-6 mol/L dihydroethidium (Sigma) in PBS buffer (pH 7.4) and incubated at 37°C for 30 minutes in a dark humidified container. Fluorescence in vessel sections was then detected by a Zeiss 210 confocal microscope with a 590-nm long-pass filter. Images of the vessels that were treated with saline were measured first. After the basal settings of the confocal microscope were adjusted, images of the treated vessels were collected digitally.

Statistical Analysis
Data were expressed as mean±SEM. Repeated-measures ANOVA was used for comparison of multiple values obtained from the same subject, whereas factorial ANOVA was used for comparing data obtained from 2 independent samples of subjects. The Bonferroni procedure was used to control type I error when needed. Significance was established at P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Arterial VCAM-1 Expression in DOCA-Salt Hypertension
There was a significant elevation of average systolic blood pressure in DOCA-salt rats compared with the sham-operated rats (115±1 versus 170±3 mm Hg for sham-operated group versus DOCA-salt group, respectively [P<0.001]; n=42 each). VCAM-1 immunoreactivity was mainly localized in the intima and the intima-media border of the vessels in DOCA-salt rats (Figure 1; sections are typical of 5 separate experiments). The difference in VCAM-1 expression of fresh isolated vessels between the DOCA-salt and sham-operated groups was determined quantitatively by ELISA and Western blotting, showing that the VCAM-1 level was significantly increased in carotid and femoral arteries from the DOCA-salt group (Figure 2; P<0.05 and P<0.01 versus sham-operated group, ELISA [n=5 to 9] and Western blot [n=3]).

View larger version (119K): [in this window] [in a new window]   Figure 1. Immunohistochemical detection of arterial VCAM-1 expression in the cross sections of a typical sham-operated rat (A and C) and DOCA-salt hypertensive rat (B and D). A and B, Carotid arteries. C and D, Femoral arteries. Positive VCAM-1 immunoreactivity, as indicated by dark brown staining, is mainly localized to the intima and intima-media border. The sections shown are typical of 5 separate experiments. Bar=0.1 mm.

View larger version (17K): [in this window] [in a new window]   Figure 2. Quantitative analysis of VCAM-1 in rat carotid and femoral arteries by ELISA (A) and Western blot (B). OD indicates optical density. A, *P<0.05 for DOCA-salt vs sham-operated groups (n=5 to 9 rats). B, Molecular band size of VCAM-1 is 100 kDa. Equal amounts of protein (10 µg) were used for each group. Cumulative densitometry was used for protein quantification with NIH image software (Scion Image). The protein level in the sham-operated group was expressed as 100%. *P<0.05 and **P<0.01 for DOCA-salt vs sham-operated groups (n=3 rats in each group).

Endogenous eNOS and MnSOD Levels in Arteries of DOCA-Salt Hypertension
As shown in Figure 3, there was no significant difference in endogenous eNOS levels in carotid and femoral arteries between DOCA-salt and sham-operated rats. However, endogenous MnSOD levels were reduced by 60% and 40% in hypertensive carotid and femoral arteries, respectively (P<0.05 versus sham-operated group, n=4).

View larger version (21K): [in this window] [in a new window]   Figure 3. Western blot analysis of endogenous eNOS and MnSOD in rat carotid and femoral arteries. Equal amounts of protein (10 µg) were used for each group. The molecular band sizes are 135 kDa for eNOS and 50 kDa for MnSOD. Cumulative densitometry was used for protein quantification with NIH image software (Scion Image). The protein level in the sham-operated group was expressed as 100%. *P<0.05 for DOCA-salt vs sham-operated groups (n=4 rats in each group).

Arterial eNOS and MnSOD Levels After Gene Transfer
Twenty-four hours after gene transfer, total eNOS and MnSOD (endogenous plus recombinant proteins) were significantly increased in transduced carotid and femoral arteries compared with nontransduced control arteries; both were taken from DOCA-salt hypertensive rats (Figure 4; P<0.05 and P<0.01 versus control group, n=3 to 5). In MnSOD-transduced vessels, an increase in viral vector titer (ie, 5x10¹⁰ pfu/mL) resulted in a further elevation of total MnSOD, suggesting a "gene-dose" effect. Immunohistochemical staining showed that positive eNOS and MnSOD immunoreactivities were localized in adventitial and intimal layers of the transduced arteries in DOCA-salt rats compared with the intimal staining in nontransduced arteries (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 4. Western blot analysis of total eNOS and MnSOD in carotid and femoral arteries of DOCA-salt rats with or without gene transfer. The molecular band sizes are 135 kDa for eNOS and 50 kDa for MnSOD. Cumulative densitometry was used for protein quantification with NIH image software (Scion Image). The protein level in nontransduced arteries of DOCA-salt rats was expressed as 100%. AdeNOS and AdMnSOD represent adenoviral vectors encoding eNOS and MnSOD genes, respectively. *P<0.05 and **P<0.01 for transduced vs non transduced controls (n=3 to 5 rats in each group).

Gene Transfer on Superoxide Level in DOCA-Salt Hypertension
After incubation with the superoxide-sensitive dye dihydroethidium, there was a marked increase in EtBr fluorescence (ie, red color), reflecting an increase in superoxide, throughout the vessel wall of a DOCA-salt carotid artery (Figure 5A) compared with the vessel wall of a sham-operated artery (Figure 5B). Twenty-four hours after the gene transfer of eNOS (Figure 5C) or MnSOD (Figure 5D) at 5x10¹⁰ pfu/mL, EtBr fluorescence was attenuated (sections were representative of 4 separate experiments). Compared with no incubation, incubation of nontransduced vessels for 24 hours did not increase the superoxide level (data not shown). Similar results were observed in femoral arteries (data not shown).

View larger version (136K): [in this window] [in a new window]   Figure 5. Fluorescent confocal micrographs showing in situ detection of superoxide in rat carotid arteries. Arterial sections were labeled with the oxidative dye dihydroethidium, which fluoresces red when oxidized to EtBr by superoxide (see Methods). Fluorescent intensity was markedly elevated across the vessel wall in a carotid artery from a DOCA-salt rat (A), as indicated by red color, compared with a vessel from a sham-operated rat (B). Gene transfer of eNOS (C) or MnSOD (D) at 5x1010 pfu/mL markedly attenuated the fluorescence intensity in vessel sections from the DOCA-salt rat. The sections shown are typical of 4 separate experiments. Bar=0.05 mm.

Gene Transfer on VCAM-1 Expression in DOCA-Salt Hypertension
At a titer of 5x10¹⁰ pfu/mL (high titer), gene transfer of either MnSOD or eNOS significantly reduced VCAM-1 levels in the carotid and femoral arteries of DOCA-salt rats (Figure 6; P<0.05 versus nontransduced control arteries, n=3 to 5). However, at a titer of 10¹⁰ pfu/mL (low titer), only MnSOD gene transfer was able to reduce VCAM-1 expression significantly. In contrast, ß-Gal reporter gene transfer had no effect on VCAM-1 expression (P>0.05 versus nontransduced control arteries, n=3). In the arteries of sham-operated rats, VCAM-1 levels remained constant before and after the vascular gene transfer of ß-Gal, eNOS, or MnSOD (please see online data supplement at http://atvb.ahajournals.org).

View larger version (24K): [in this window] [in a new window]   Figure 6. Western blot analysis of VCAM-1 in carotid and femoral arteries of DOCA-salt rats with or without gene transfer. An equal amount of protein (10 µg) was used for each group. The molecular band size is 100 kDa for VCAM-1. Cumulative densitometry was used for protein quantification with NIH image software (Scion Image). The protein level in nontransduced control arteries was expressed as 100%. Low and high represent the titer of virus vector at 1x1010 and 5x1010 pfu/mL, respectively. *P<0.05 vs nontransduced control arteries (n=3 to 5 rats in each group).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study investigated the effect of ex vivo gene transfer of eNOS and MnSOD on the regulation of arterial VCAM-1 expression and superoxide production in a rat model of DOCA-salt hypertension. The major new findings of the present study are as follows: (1) VCAM-1 expression was significantly increased in carotid and femoral arteries of DOCA-salt hypertensive rats, (2) endogenous MnSOD, but not eNOS, was reduced with increased superoxide level in these vessels, and (3) gene transfer of MnSOD and eNOS suppressed arterial VCAM-1 expression with a concomitant reduction of superoxide in DOCA-salt hypertensive rats. These results suggest that superoxide plays an important role in stimulating vascular VCAM-1 expression in DOCA-salt hypertension and that mitochondrial superoxide may be a key source of oxidative stress. Gene transfer of MnSOD or eNOS may be a useful strategy in ameliorating the oxidative stress–induced vascular dysfunction and atherogenesis in hypertension.

Superoxide contributes to vascular dysfunction, atherogenesis, and hypertension.^13,22 In an animal model of hypertension, Tummala et al¹¹ reported that Ang II stimulates VCAM-1 expression via the activation of oxidative signaling pathways involving the redox-sensitive transcription factor nuclear factor-B (NF-B) and the upregulation of genes downstream from NF-B, including VCAM-1. Other groups have shown that Ang II stimulates superoxide production via a membrane-bound NADPH oxidase.^9,10 Thus, the renin-angiotensin system seems to play an important role in regulating vascular VCAM-1 expression via superoxide production. Recently, it has been reported that there is a remarkably high level of superoxide in the aortas of DOCA-salt hypertensive rats,^14,15 a model characterized by its depressed plasma renin activity.¹⁶ These latter studies suggest that Ang II is not a sole stimulus of superoxide and raise the possibility that the increased superoxide in DOCA-salt hypertension may contribute to vascular adhesion molecule expression, such as VCAM-1. The present study addressed such a possibility and demonstrated a significant increase of arterial VCAM-1 expression and superoxide level in DOCA-salt hypertensive rats. Our results further showed that the increase in superoxide was accompanied by a reduced endogenous MnSOD level in carotid and femoral arteries, 2 different vessel types that are prone to the development of atherosclerosis in hypertensive patients. These observations suggest that in addition to Ang II–induced hypertension, superoxide may also contribute to the enhanced arterial VCAM-1 expression in salt-sensitive hypertension. This assumption is further supported by our findings that MnSOD gene transfer to these vessels attenuated VCAM-1 expression and superoxide level. Therefore, superoxide-induced oxidative stress may be a general stimulus for VCAM-1 expression in hypertension associated with either a high or low renin-angiotensin state. In addition, the observed increase in superoxide levels may not be related to the stimulation of NADPH oxidase by Ang II, inasmuch as this model of hypertension is well known for its suppressed plasma renin and Ang II levels.¹⁶

NO possesses important antihypertensive and antiatherogenic effects, including vasodilatation,²² inhibition of VCAM-1 expression,⁷ NF-B activation,²³ platelet adhesion, and smooth muscle proliferation.²² NO-dependent vasodilatation is markedly impaired in atherosclerosis and hypertension.²² In patients with essential hypertension or salt-sensitive hypertension, agonist-induced NO release in the forearm is attenuated.^24–26 A major mechanism responsible for such an impairment is superoxide generated in the presence of the disease, which inactivates NO rapidly.^13,22 In various atherogenic models, the accumulation of T lymphocytes and monocytes at sites of vascular inflammation was shown to be VCAM-1 dependent.^2–5,27 Inflammatory mediators, including NF-B, are activated in part through the stimulation of superoxide production, resulting in attraction of macrophages, stimulation of foam cell formation, inflammation, and atherogenesis.^2–5,27

Because of the pivotal role of NO in vascular protection, we hypothesized that gene transfer of eNOS would be able to inhibit VCAM-1 expression, either directly or by neutralizing the effect of superoxide. We found that eNOS gene transfer was able to reduce the arterial VCAM-1 level by 50% and, at the same time, to attenuate the superoxide level in DOCA-salt hypertensive rats. These findings were consistent with an earlier study²⁸ in atherosclerotic carotid arteries of the cholesterol-fed rabbits in which VCAM-1 expression and inflammatory cell infiltration were effectively ameliorated by neuronal NO synthase gene transfer.

The results of our eNOS gene transfer experiments seemed to be contrary to the conventional assumption that the generation of more NO would be inactivated by superoxide. However, these findings were in agreement with recent studies that have convincingly demonstrated that ex vivo and in vivo gene transfer of eNOS or neuronal NO synthase restores NO-mediated arterial relaxations that were impaired by increased superoxide production in hypertensive,^29,30 atherosclerotic,^31–34 or diabetic^35–38 animals. These experimental observations support the novel concept that NO generated by recombinant NO synthase, as a result of gene transfer, provides an effective means of inactivating superoxide and, thereby, improving vascular function, including endothelium-dependent relaxation, in vessels with elevated superoxide.³⁹

In the present study, we found that compared with eNOS gene transfer, MnSOD gene transfer is more effective in suppressing arterial VCAM-1 expression. Our data showed that at a vector titer of 5x10¹⁰ pfu/mL, eNOS gene transfer decreased VCAM-1 levels by 50%, an effect that was achieved with MnSOD gene transfer at a titer 5-fold lower. When the titer was increased to the same as used for eNOS, gene transfer of MnSOD reduced the VCAM-1 level further (to up to 70%). The difference in the efficacy of MnSOD and eNOS gene transfer may be explained in part by our findings that endogenous MnSOD, but not eNOS, was significantly reduced in the hypertensive vessels. Accordingly, MnSOD gene transfer may restore the functional capacity of the antioxidant enzyme in scavenging elevated superoxide. In contrast, a larger amount of NO may be required from eNOS gene transfer to neutralize the stimulatory effect of superoxide on VCAM-1 and directly inhibit its expression.

It is interesting to notice that although recombinant eNOS and MnSOD were expressed only in the endothelium and adventitia after gene transfer, they were effective in reducing the increased superoxide observed throughout the vessel wall in DOCA-salt hypertensive rats. Although the exact mechanisms for these experimental observations remain to be determined, we speculate that the NO generated by eNOS gene transfer, in either the endothelium or adventitia, may neutralize superoxide in the adjacent smooth muscle layer because NO is highly diffusible. This speculation is consistent with a previously published study²⁹ in which gene transfer of eNOS resulted in a significant improvement of Ang II–induced impairment of endothelium-dependent relaxations to acetylcholine, despite increased superoxide levels across the vessel wall. Although recombinant superoxide dismutase (SOD) can scavenge superoxide in endothelial and adventitial layers because of its large molecular weight, this may not be the case inside the smooth muscle cells. However, because superoxide has been shown to cross erythrocyte⁴⁰ and endothelial cell⁴¹ membranes via anion channels (eg, chloride channels), we speculate that it may diffuse outward into the lumen and perivascular site because of its high level in smooth muscle and relative low levels in the endothelium and adventitia as it is being scavenged at both sites after gene transfer. Thus, overexpression of MnSOD in the endothelium and adventitia may produce a "diffusion-gradient" effect through which superoxide gets into the 2 outside layers, whereby it is scavenged.

Previous studies have reported that vascular gene transfer of CuZnSOD or extracellular SOD did not improve superoxide-impaired endothelium-dependent relaxation in animal models of Ang II–induced hypertension,²⁹ atherosclerosis,¹² or diabetes.³³ On the other hand, studies from other groups have shown that gene transfer of MnSOD and CuZnSOD normalized such impaired vasomotor function in diabetic vessels³⁷ and atherosclerotic vessels without plaque formation.³⁸ On the basis of the different results of these studies, we decided to examine the effect of MnSOD gene transfer, because mitochondria may also be a key source of superoxide production in addition to the cytosol and extracellular space.¹³ Our results of MnSOD gene transfer on vascular VCAM-1 and superoxide levels are in agreement with the latter reports. Although the exact reasons are unclear for these different experimental observations, the source of superoxide production (eg, cytosolic versus mitochondrial), the disease model studied (eg, Ang II versus DOCA-salt hypertension), the stages of disease examined (eg, vessels before and after atherosclerotic plaque formation), and the animal species used (eg, rabbit versus rat) are factors to be considered to explain such discrepancies. In addition, the effect of MnSOD gene transfer on NO-mediated endothelium-dependent relaxation in DOCA-salt hypertension remains to be determined.

In summary, the results of the present study demonstrate that in the carotid and femoral arteries of DOCA-salt hypertensive rats, there was a significant increase in VCAM-1 expression and superoxide level. Our data have also shown, for the first time, that the endogenous MnSOD level was decreased in DOCA-salt hypertension. Finally, gene transfer of MnSOD and eNOS was effective in reducing VCAM-1 and superoxide levels in a model of hypertension associated with a low renin state. Gene therapy strategies aimed at reducing superoxide-induced oxidative stress may be useful in ameliorating vascular dysfunction and atherogenesis in hypertension.⁴²

	Acknowledgments

 
This work was supported by American Heart Association/Midwest Affiliate Scientist Development Grant 0130537Z, American Diabetes Association Regular Research Award 7-01-RA-10, Juvenile Diabetes Research Foundation Innovative Grant 5-2001-311, a Michigan State University Foundation Capacity Building Grant (all to Dr Chen), and a Program Project Grant (No. 1592) of the Michigan Life Sciences Corridor Fund (to Drs Wang and Chen).

Received October 23, 2001; accepted November 27, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233–241.[CrossRef][Medline] [Order article via Infotrieve]

2. Krieglstein CF, Granger DN. Adhesion molecules and their role in vascular disease. Am J Hypertens. 2001; 14: 44S–54S.[CrossRef][Medline] [Order article via Infotrieve]

3. Price DT, Loscalzo J. Cellular adhesion molecules and atherogenesis. Am J Med. 1999; 107: 85–97.[Medline] [Order article via Infotrieve]

4. Chia MC. The role of adhesion molecules in atherosclerosis. Crit Rev Clin Lab Sci. 1998; 35: 573–602.[CrossRef][Medline] [Order article via Infotrieve]

5. Ley K, Huo Y. VCAM-1 is critical in atherosclerosis. J Clin Invest. 2001; 107: 1209–1210.[Medline] [Order article via Infotrieve]

6. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V, Gutierrez-Ramos J-C, Connelly PW, Milstone DS. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest. 2001; 107: 1255–1262.[Medline] [Order article via Infotrieve]

7. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA Jr, Shin WS, Liao JK. Nitric oxide decreased cytokine-induced endothelial activation: nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest. 1995; 96: 60–68.[Medline] [Order article via Infotrieve]

8. Artigues C, Richard V, Roussel C, Lallemand F, Henry J-P, Thuillez C. Increased endothelium-monocyte interactions in salt-sensitive hypertension: effect of L-arginine. J Cardiovasc Pharmacol. 2000; 35: 468–473.[CrossRef][Medline] [Order article via Infotrieve]

9. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADP and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.[Abstract/Free Full Text]

10. Pagano PJ, Clark JK, Cifuentes-Pagano ME, Clark SM, Callis GM, Quinn MT. Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. Proc Natl Acad Sci U S A. 1997; 94: 14483–14488.[Abstract/Free Full Text]

11. Tummala PE, Chen XL, Sundell CL, Laursen JB, Hammes CP, Alexander RW, Harrison DG, Medford RM. Angiotensin II induces vascular cell adhesion molecule-1 expression in rat vasculature: a potential link between the renin-angiotensin system and atherosclerosis. Circulation. 1999; 100: 1223–1229.[Abstract/Free Full Text]

12. Miller FJ, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.[Abstract/Free Full Text]

13. McIntyre M, Bohr DF, Dominiczak AF. Endothelium function in hypertension: the role of superoxide anion. Hypertension. 1999; 34: 539–545.[Abstract/Free Full Text]

14. Somers MJ, Mavromatis K, Galis ZS, Harrison DG. Vascular superoxide production and vasomotor function in hypertension induced by deoxycorticosterone acetate-salt. Circulation. 2000; 101: 1722–1728.[Abstract/Free Full Text]

15. Wu R, Millette E, Wu L, Champlain JD. Enhanced superoxide anion formation in vascular tissues from spontaneously hypertensive and desoxycorticosterone acetate-salt hypertensive rats. J Hypertens. 2001; 19: 741–748.[CrossRef][Medline] [Order article via Infotrieve]

16. Gavras H, Brunner HR, Laragh JH, Vaughan ED Jr, Koss M, Cote LJ, Gavras I. Malignant hypertension resulting from deoxycorticosterone acetate and salt excess: role of renin and sodium in vascular changes. Circ Res. 1975; 36: 300–309.[Abstract/Free Full Text]

17. Watts SW, Fink GD. 5-HT_2B-receptor antagonist LY-272015 is antihypertensive in DOCA-salt-hypertensive rats. Am J Physiol. 1999; 276: H944–H952.[Medline] [Order article via Infotrieve]

18. Chen AF, O’Brien T, Tsutsui M, Kinoshita H, Pompili VJ, Crotty TB, Spector DJ, Katusic ZS. Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery. Circ Res. 1997; 80: 327–335.[Abstract/Free Full Text]

19. Tsutsui M, Chen AF, O’Brien T, Crotty TB, Katusic ZS. Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium. Arterioscler Thromb Vasc Biol. 1998; 18: 1231–1241.[Abstract/Free Full Text]

20. Chen AF, Jiang S, Crotty TB, Tsutsui M, Smith LA, O’Brien T, Katusic ZS. Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries. Proc Natl Acad Sci U S A. 1997; 94: 12568–12573.[Abstract/Free Full Text]

21. Li LX, Chen JX, Liao DF, Yu L. Probucol inhibits oxidized-low density lipoprotein-induced adhesion of monocytes to endothelial cells by reducing P-selectin synthesis in vitro. Endothelium. 1998; 6: 1–8.[Medline] [Order article via Infotrieve]

22. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.[Abstract/Free Full Text]

23. Wee Soo S, Hong YH, Peng HB, De Caterina R, Libby P, Liao JK. Nitric oxide attenuates vascular smooth muscle cell activation by interferongamma: the role of constitutive NF-kappaB activity. J Biol Chem. 1996; 271: 11317–11324.[Abstract/Free Full Text]

24. Panza JA, Quyyumi AA, Brush JE Jr, Epstein SE. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med. 1990; 323: 22–27.[Abstract]

25. Linder L, Kiowski W, Buhler FR, Luscher TF. Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo blunted response in essential hypertension. Circulation. 1990; 81: 1762–1767.[Abstract/Free Full Text]

26. Ghiadoni L, Virdis A, Taddei S, Gonzales J, Salazar J. Defective nitric oxide pathway in salt-sensitive essential hypertensive patients. Am J Hypertens. 1997; 10: 20A.Abstract.[CrossRef]

27. Pueyo ME, Gonzalez W, Nicoletti A, Savoie F, Arnal JF, Michel JB. Angiotensin II stimulates endothelial vascular cell adhesion molecule-1 via nuclear factor-B activation induced by intracellular oxidative stress. Arterioscler Thromb Vasc Biol. 2000; 20: 645–651.[Abstract/Free Full Text]

28. Qian H, Neplioueva V, Shetty GA, Channon KM, George SE. Nitric oxide synthase gene therapy rapidly reduces adhesion molecule expression and inflammatory cell infiltration in carotid arteries of cholesterol-fed rabbits. Circulation. 1999; 99: 2979–2982.[Abstract/Free Full Text]

29. Nakane H, Miller FJ Jr, Faraci FM, Toyoda K, Heistad DD. Gene transfer of endothelial nitric oxide synthase reduces angiotensin II-induced endothelial dysfunction. Hypertension. 2000; 35: 595–601.[Abstract/Free Full Text]

30. Alexander MY, Brosnan MJ, Hamilton CA, Fennell JP, Beattie EC, Jardine E, Heistad DD, Dominiczak AF. Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP. Cardiovasc Res. 2000; 47: 609–617.[Abstract/Free Full Text]

31. Ooboshi H, Toyoda K, Faraci FM, Lang MG, Heistad DD. Improvement of relaxation in an atherosclerotic artery by gene transfer of endothelial nitric oxide synthase. Arterioscler Thromb Vasc Biol. 1998; 18: 1752–1756.[Abstract/Free Full Text]

32. Channon KM, Qian H, Neplioueva V, Blazing MA, Olmez E, Shetty GA, Youngblood SA, Pawloski J, McMahon T, Stamler JS, et al. In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-fed rabbits. Circulation. 1998; 98: 1905–1911.[Abstract/Free Full Text]

33. Mozes G, Kullo IJ, Mohacsi TG, Cable DG, Spector DJ, Crotty TB, Gloviczki P, Katusic ZS, O’Brien T. Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine. Atherosclerosis. 1998; 141: 265–271.[CrossRef][Medline] [Order article via Infotrieve]

34. Sato J, Mohacsi T, Noel A, Jost C, Gloviczki P, Mozes G, Katusic ZS, O’Brien T. In vivo gene transfer of endothelial nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits enhances endothelium-dependent relaxations. Stroke. 2000; 31: 968–975.[Abstract/Free Full Text]

35. Lund DD, Faraci FM, Miller FJ Jr, Heistad DD. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation. 2000; 101: 1027–1033.[Abstract/Free Full Text]

36. Zanetti M, Sato J, Katusic ZS, O’Brien T. Gene transfer of endothelial nitric oxide synthase alters endothelium-dependent relaxations in aortas from diabetic rabbits. Diabetologia. 2000; 43: 340–347.[CrossRef][Medline] [Order article via Infotrieve]

37. Zanetti M, Sato J, Katusic ZS, O’Brien T. Gene transfer of superoxide dismutase isoforms reverses endothelial dysfunction in diabetic rabbit aorta. Am J Physiol. 2001; 280: H2516–H2523.

38. Zanetti M, Sato J, Jost CJ, Gloviczki P, Katusic ZS, O’Brien T. Gene transfer of manganese superoxide dismutase reverses vascular dysfunction in the absence but not in the presence of atherosclerotic plaque. Hum Gene Ther. 2001; 12: 1407–1416.[CrossRef][Medline] [Order article via Infotrieve]

39. Heistad DD. Gene transfer to blood vessels: a research tool and potential therapy. Am J Hypertens. 2001; 14: 28S–32S.[CrossRef][Medline] [Order article via Infotrieve]

40. Lynch RE, Fridovich I. Permeation of the erythrocyte stroma by superoxide radical. J Biol Chem. 1978; 253: 4697–4699.[Abstract/Free Full Text]

41. Brzezinska AK, McLeod J, Chilian WM. Chloride channels conduct superoxide (O₂^- · ) radical across the plasma membrane of vascular endothelial cells. Circulation. 2001; 104 (suppl II): II-99.Abstract.

42. Chen AF, O’Brien T, Katusic ZS. Functional influence of gene transfer of recombinant nitric oxide synthase to vascular tissue.In: Ignarro LJ, ed. Nitric Oxide: Biology and Pathobiology. San Diego, Calif: Academic Press; 2000: 525–545.

This article has been cited by other articles:

				L. Li and B. Frei Prolonged Exposure to LPS Increases Iron, Heme, and p22phox Levels and NADPH Oxidase Activity in Human Aortic Endothelial Cells: Inhibition by Desferrioxamine Arterioscler Thromb Vasc Biol, May 1, 2009; 29(5): 732 - 738. [Abstract] [Full Text] [PDF]

				K. A. Nath, L. V. d'Uscio, J. P. Juncos, A. J. Croatt, M. C. Manriquez, S. T. Pittock, and Z. S. Katusic An analysis of the DOCA-salt model of hypertension in HO-1-/- mice and the Gunn rat Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H333 - H342. [Abstract] [Full Text] [PDF]

				L. Li and B. Frei Iron Chelation Inhibits NF-{kappa}B-Mediated Adhesion Molecule Expression by Inhibiting p22phox Protein Expression and NADPH Oxidase Activity Arterioscler Thromb Vasc Biol, December 1, 2006; 26(12): 2638 - 2643. [Abstract] [Full Text] [PDF]

				D.-D. Chen and A. F. Chen CuZn Superoxide Dismutase Deficiency: Culprit of Accelerated Vascular Aging Process Hypertension, December 1, 2006; 48(6): 1026 - 1028. [Full Text] [PDF]

				H. Hong, J.-S. Zeng, D. L. Kreulen, D. I. Kaufman, and A. F. Chen Atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in ischemic stroke Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2210 - H2215. [Abstract] [Full Text] [PDF]

				C. Zhang, R. Lopez-Ridaura, D. J. Hunter, N. Rifai, and F. B. Hu Common variants of the endothelial nitric oxide synthase gene and the risk of coronary heart disease among u.s. Diabetic men. Diabetes, July 1, 2006; 55(7): 2140 - 2147. [Abstract] [Full Text] [PDF]

				C. Schwencke, R. C. Braun-Dullaeus, C. Wunderlich, and R. H. Strasser Caveolae and caveolin in transmembrane signaling: Implications for human disease Cardiovasc Res, April 1, 2006; 70(1): 42 - 49. [Abstract] [Full Text] [PDF]

				J.-D. Luo, Y.-Y. Wang, W.-L. Fu, J. Wu, and A. F. Chen Gene Therapy of Endothelial Nitric Oxide Synthase and Manganese Superoxide Dismutase Restores Delayed Wound Healing in Type 1 Diabetic Mice Circulation, October 19, 2004; 110(16): 2484 - 2493. [Abstract] [Full Text] [PDF]

				J. Segui, M. Gironella, M. Sans, S. Granell, F. Gil, M. Gimeno, P. Coronel, J. M. Pique, and J. Panes Superoxide dismutase ameliorates TNBS-induced colitis by reducing oxidative stress, adhesion molecule expression, and leukocyte recruitment into the inflamed intestine J. Leukoc. Biol., September 1, 2004; 76(3): 537 - 544. [Abstract] [Full Text] [PDF]

				L. Li, Y. Chu, G. D. Fink, J. F. Engelhardt, D. D. Heistad, and A. F. Chen Endothelin-1 Stimulates Arterial VCAM-1 Expression Via NADPH Oxidase-Derived Superoxide in Mineralocorticoid Hypertension Hypertension, November 1, 2003; 42(5): 997 - 1003. [Abstract] [Full Text] [PDF]

				N. Kobayashi, S.-i. Mita, K. Yoshida, T. Honda, T. Kobayashi, K. Hara, S. Nakano, Y. Tsubokou, and H. Matsuoka Celiprolol Activates eNOS Through the PI3K-Akt Pathway and Inhibits VCAM-1 Via NF-{kappa}B Induced by Oxidative Stress Hypertension, November 1, 2003; 42(5): 1004 - 1013. [Abstract] [Full Text] [PDF]

				J.-S. Zheng, X.-Q. Yang, K. J. Lookingland, G. D. Fink, C. Hesslinger, G. Kapatos, I. Kovesdi, and A. F. Chen Gene Transfer of Human Guanosine 5'-Triphosphate Cyclohydrolase I Restores Vascular Tetrahydrobiopterin Level and Endothelial Function in Low Renin Hypertension Circulation, September 9, 2003; 108(10): 1238 - 1245. [Abstract] [Full Text] [PDF]

				L. Li, S. W. Watts, A. K. Banes, J. J. Galligan, G. D. Fink, and A. F. Chen NADPH Oxidase-Derived Superoxide Augments Endothelin-1-Induced Venoconstriction in Mineralocorticoid Hypertension Hypertension, September 1, 2003; 42(3): 316 - 321. [Abstract] [Full Text] [PDF]

				M. Christofidou-Solomidou, A. Scherpereel, R. Wiewrodt, K. Ng, T. Sweitzer, E. Arguiri, V. Shuvaev, C. C. Solomides, S. M. Albelda, and V. R. Muzykantov PECAM-directed delivery of catalase to endothelium protects against pulmonary vascular oxidative stress Am J Physiol Lung Cell Mol Physiol, August 1, 2003; 285(2): L283 - L292. [Abstract] [Full Text] [PDF]

				L. Li, J. J. Galligan, G. D. Fink, and A. F. Chen Vasopressin Induces Vascular Superoxide Via Endothelin-1 in Mineralocorticoid Hypertension Hypertension, March 1, 2003; 41(3): 663 - 668. [Abstract] [Full Text] [PDF]

				L. Li, G. D. Fink, S. W. Watts, C. A. Northcott, J. J. Galligan, P. J. Pagano, and A. F. Chen Endothelin-1 Increases Vascular Superoxide via EndothelinA-NADPH Oxidase Pathway in Low-Renin Hypertension Circulation, February 25, 2003; 107(7): 1053 - 1058. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, L. Articles by Chen, A. F. Search for Related Content PubMed PubMed Citation Articles by Li, L. Articles by Chen, A. F. Related Collections Hypertension - basic studies Gene therapy Oxidant stress Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors