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Cardiovascular Research 2002 53(4):1010-1018; doi:10.1016/S0008-6363(01)00535-1
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Copyright © 2002, European Society of Cardiology

Chronic antioxidant supplementation attenuates nuclear factor-{kappa}B activation and preserves endothelial function in hypercholesterolemic pigs

Martin Rodriguez-Porcela, Lilach O Lermanb, David R Holmes, Jr.a, Darcy Richardsona, Claudio Napolic,d and Amir Lermana,*

aDepartment of Internal Medicine, Division of Cardiovascular Diseases, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
bDepartment of Internal Medicine, Division of Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
cDepartment of Medicine, University of Naples, Naples, Italy
dDepartment of Medicine—0682, University of California, San Diego, CA, USA

* Corresponding author. Tel.: +1-507-255-4152; fax: +1-507-255-2550 lerman.amir{at}mayo.edu

Received 3 July 2001; accepted 1 November 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Hypercholesterolemia (HC), a pro-oxidant condition, activates nuclear factor-kappa beta (NF-{kappa}B) and is associated with coronary endothelial dysfunction. The physiological significance of in vivo chronic antioxidant intervention on HC-induced NF-{kappa}B activation and coronary endothelial function remains unclear. Methods: Four groups of pigs were studied after 12 weeks of normal diet, normal diet with concomitant antioxidant intervention (100 IU/kg of vitamin E and 1 g of vitamin C daily), 2% HC diet, or HC diet+antioxidant supplementation. NF-{kappa}B activation and the nitric oxide (NO) pathway were investigated by Western blotting and immunohistochemistry, while oxidative stress was evaluated by coronary artery tissue radical scavenger activity and levels of vitamin E and C. Endothelial function was studied in vitro by coronary vasoreactivity to bradykinin and substance P. Results: HC animals had increased activation of NF-{kappa}B, decreased endothelial NO synthase expression, and decreased radical scavenger system activity, associated with impaired coronary endothelial function. Antioxidant supplementation in HC normalized NF-{kappa}B activation and NO bioactivity, and preserves coronary endothelial function. Conclusions: This study demonstrates for the first time that in vivo chronic interruption of the endogenous oxidative stress cascade reduces HC-induced NF-{kappa}B activation and normalizes NO bioactivity in association with preservation of coronary endothelial function. This study suggests a role for increased oxidative stress and NF{kappa}B activation in early atherosclerosis.

KEYWORDS Atherosclerosis; Coronary circulation; Endothelial function; Free radicals


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Experimental hypercholesterolemia (HC) is associated with increased production of reactive oxygen species (ROS) and other radicals [1], a state known as increased oxidative stress. Increased oxidative stress in the arterial wall may also be characterized by decreased activity of endogenous cellular radical scavengers [2], increased production of vasoconstrictors [3], and decreased bioavailability of nitric oxide (NO) [4]. NO is involved in numerous antiatherogenic signaling pathways and transcriptional events in the vascular wall, and a decrease in its bioavailability may impair regulation of endothelial function and may allow activation of intra-cellular mechanisms that can ultimately lead to atherosclerotic lesion formation.

Nuclear factor-{kappa}B (NF-{kappa}B) is a transcription factor that is involved in the regulation of many cellular target genes [5]. Activated NF{kappa}B binds to the specific promoter regions of target genes such as IL-1, VCAM, and ICAM and many others which are involved in the progression of atherosclerosis [6,7]. Furthermore, activation of NF{kappa}B has been related to NO bioavailability [8] and ROS production [5]. While ROS contribute to NF{kappa}B activation [5], an intact NO pathway system stabilizes it and prevents its activation [9]. Thus, a balance between oxidative status and NO-dependent pathways may be one of the regulatory mechanisms of NF{kappa}B activation.

We have previously demonstrated that experimental hypercholesterolemia in the pig was associated with activation of NF-{kappa}B in the arterial wall [8], as well as endothelial dysfunction [8,10], and a decrease in the endogenous NO bioavailability in the coronary vasculature [11]. More recently, we reported that chronic concomitant administration of antioxidant vitamins E and C decreased endogenous oxidative stress in the myocardial tissue and preserved myocardial perfusion in experimental hypercholesterolemia in vivo [12]. Previous studies in vitro have demonstrated that antioxidants may attenuate NF{kappa}B activation [5]. However, there is a void of information regarding the potential of chronic attenuation of endogenous oxidative stress in vivo to modulate both the NO cascade and activation of NF-{kappa}B. Furthermore, the functional significance of this effect remains unclear.

Thus, the present study was designed to test the hypothesis that chronic attenuation of the oxidative stress cascade would minimize coronary NF-{kappa}B activation in conjunction with preservation of coronary endothelial function in experimental hypercholesterolemia in the pig.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal preparation
All procedures using animals were reviewed and approved by the Mayo Foundation Institutional Animal Care and Use Committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Four groups of female juvenile domestic crossbred pigs (50–55 kg) were studied after 12 weeks of diet. Group 1 (n=10) were fed a normal diet, while group 3 (HC, n=8) was placed on an atherogenic diet of 2% cholesterol and 15% lard by weight (TD 93296, Harlan Teklad, Madison, WI) [12]. Groups 2 (n=6) and 4 (n=6) received normal and HC diet, respectively, and in addition were placed on daily antioxidant combination supplementation, consisting of vitamin E (100 IU/kg/day) and vitamin C (1000 mg/day) (normal+anti-ox and HC+anti-ox, respectively). A combination of antioxidants vitamins E and C has been previously shown to have a synergistic effect in blocking the oxidative pathway [13,14].

Plasma lipid profiles (Roche, Nutley, NJ) were determined after 12 weeks of diet in all four groups. Following completion of diet, euthanasia was performed by an intravenous administration of 30 mg/kg pentobarbital sodium (Sleepaway®, Fort Dodge Laboratories, IA), typically 12 h after last feeding, and tissue was harvested for in vitro studies. These included assessment of the expression of NF-{kappa}B and endothelial nitric oxide synthase (eNOS), redox status and NO pathway functionality.

2.2. NF{kappa}B and eNOS
2.2.1. Western blotting
Samples of epicardial coronary artery from each of the four groups were freshly frozen in liquid nitrogen and subsequently homogenized in lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, PMSF 100 µg/ml, aprotinin 1 µg/ml, 1% NP-40, 0.5% sodium deoxycholate) using a tissue homogenizer as was previously described [8]. The lysate was analyzed for protein content using a Bradford assay (Bio-Rad, CA, USA). Equal amounts of protein were resolved under reducing conditions on an 8% SDS–polyacrylamide gel. Immunoblotting was performed using monoclonal antibodies for NF-{kappa}B (Boehringer-Mannheim, IN, USA) at a dilution of 1:100 and eNOS (Transduction Laboratories, Lexington, KY) at a dilution of 1:1000 in a non-fat milk/Tris buffer. The membranes were subsequently probed with a secondary anti-mouse antibody conjugated to horseradish peroxidase (Amersham Life Sciences, IL, USA) at a dilution of 1:5000 and developed with chemiluminescence (Pierce, IL, USA) [8]. Membranes were then exposed to X-ray film (Kodak, NY, USA) which was subsequently developed. Equal protein loading was confirmed with Ponceau staining. Densitometry was performed using Scion Imagine and densitometric units measured, as described [8].

2.2.2. Immunohistochemistry
Immunohistochemistry was performed as previously described [8,10]. Briefly, frozen sections of epicardial coronary artery from animals of each of the four groups were cut in 5-µm cross-sections and mounted on positively charged slides. The slides were air dried for 30 min. Endogenous peroxidase activity was blocked by placing the slides in 1.5% hydrogen peroxide and 50% absolute methanol for 10 min and then the slides were rinsed. The slides were pretreated with 0.25% sodium dodecyl sulfate for 10 min. To block nonspecific binding sites, the tissue was incubated with 5% goat serum (Dako, Carpineria, CA)/phosphate-buffered saline/Tween 20 for 10 min. Antibodies to the activated p65 subunit of NF-{kappa}B (Boehringer-Mannheim, IN, USA, 1:25 dilution of 1 mg/ml of antibody) [8] and endothelial NO synthase (eNOS) (Transduction Laboratories, Lexington, KY, 1:400 dilution of 250 mg/ml of antibody) [10] were then added and incubated overnight at 4 °C. The slides were rinsed and then incubated for 30 min with biotinylated goat anti-mouse IgG diluted 1:400 and 2% normal swine serum, rinsed again, incubated with peroxidase-labeled streptavidin diluted 1:500 for 30 min, and rinsed. Next, the tissue was stained for 15 min with 3-amino-9-ethylcarbazole solution and rinsed. To enhance nuclear detail, the slides were counterstained with hematoxylin [8,10].

2.3. Redox status
Vitamins E ({alpha}-tocopherol) and C (ascorbic acid) concentration in coronary artery tissue was determined by HPLC [12]. Tissue concentrations of oxygen-radical scavengers were determined spectophotometrically. Homogenates in potassium phosphate buffer, pH 7.4, containing 10 mol/l deferoxamine, 0.03% butylated hydroxytoluene, and 2% ethanol equilibrated with nitrogen (to reduce auto-oxidation) were centrifuged at 1000xg for 15 min at 4 °C to remove nuclei and tissue debris. The supernatant was centrifuged again at 30 000xg for 35 min at 4 °C. Glutathione peroxidase, catalase, and both the copper–zinc and manganese forms of superoxide dismutase (CuZn- and Mn-SOD, respectively) activities were determined spectrophotometrically, as previously described [12]. All enzyme activities were normalized for protein content [15].

2.4. Coronary artery smooth muscle cyclic GMP production
To assess the NO pathway activity, cGMP production was measured in smooth muscle cells in response to exogenous NO donation as previously described [16]. Briefly, in vascular rings from each group the endothelium was carefully removed and the rings were placed in an organ chamber filled with Krebs solution. After 1 h of incubation, 146 µl of 3-isobuthyl-1-methyl-xanthine 10–4 mol/l (to inhibit the cAMP phosphodiesterase) and 100 µl of indomethacin 10–5 mol/l (to block the production of prostaglandins and focus on the NO pathway) were added to the solution in the organ chamber for 30 min. Later, samples were randomized to either standards (controls) or treated with diethylamine (a NO donor, [16]) 10–6 mol/l for 1 min and all samples were then shock-frozen. Samples were then prepared as previously described [16], mounted on the scintillator counter and subsequently tested.

2.5. Vascular endothelial function
2.5.1. Epicardial vessels
Arterial rings were prepared as previously described [10,17]. Briefly, hearts from the four groups were placed into cold modified Krebs–Ringer bicarbonate solution (control solution). Rings of tissue 2–3 mm long were dissected, transferred to organ chambers with 25 ml of control solution and oxygenated with 94% O2 and 6% CO2. The tissue was suspended between two stirrups and connected to a strain gauge for continuous recording of isometric tension. The artery rings were equilibrated for 1 h at a resting tension. Viability of the vessels was confirmed by a contractile response to 20 mmol/l KCl at baseline, at 2, 4, and 6 g of tension, each time after the KCl had been washed out. At 6 g of tension, all vessels were exposed to substance P (10–6 mol/l; Sigma, St. Louis, MO), an endothelium-dependent vasodilator, to verify the functional integrity of the vascular endothelium. All chambers were then washed out using the control solution.

After an equilibrium period of 30 min, rings were pre-contracted with 10–7 mol/l endothelin-1 (Pheonix Pharmaceuticals, Mountain View, CA), an effective coronary vasoconstrictor and then the response to 10–11–10–6.5 mol/l bradykinin (Sigma) was obtained. In additional vascular rings from each group, a dose response to 10–11–10–6.5 mol/l of substance P was obtained. Complete relaxation of each ring was tested by exposure to 10–3.5 mol/l papaverine.

2.5.2. Arterioles
Coronary vasomotor tone in the microvessels was determined using previously described methods [18]. Briefly, segments 2–3 mm long of secondary branch of the left circumflex artery which were less than 500 µm in diameter were dissected, transferred to an arteriograph, and then mounted onto microcannullas (Living System Instrumentation, Burlington, VT). The arteriograph was placed on a microscope (Diaphot-TMD, Nikon, Japan) which had a video camera connected to the viewing tube. The signal obtained was electronically processed, and both the inner diameter (lumen) and wall thickness measured and recorded.

Vessels were pre-contracted with 10–8 mol/l endothelin-1 and the response to bradykinin (10–11–10–6) mol/l was then recorded. Complete relaxation of each arteriole was obtained by exposure to 10–4 mol/l papaverine (Sigma).

2.6. Statistical analysis
Data are expressed as mean±S.E.M. (mean arterial pressure, heart rate, Western blotting, and cGMP production) or as percent change from the maximal contraction (in vitro vascular reactivity). Within each group, repeated measurements were analyzed with repeated measures ANOVA followed by the Bonferroni t-test, or by unpaired Student's t-test between groups. Statistical significance was accepted for a P value of <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Serum cholesterol and systemic hemodynamics
After 12 weeks of HC diet, total serum cholesterol levels in both HC groups were significantly and similarly higher than in normal pigs (treated or untreated, Table 1). This was associated with a significant increase in HDL and LDL, whereas triglycerides remained unchanged. There was no difference in cholesterol levels (total, HDL, triglycerides or LDL) between HC pigs and HC pigs treated with antioxidants (Table 1), signifying that antioxidant supplementation had no effect on cholesterol levels. There were no significant differences in mean arterial pressure or heart rate among the four groups (Table 1).


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  		Table 1 Lipid profiles (total, HDL, LDL cholesterol, and triglycerides, expressed in mg/dl and mmol/l), mean arterial pressure (MAP, mmHg), and heart rate (HR, bpm) in normal, normal treated with antioxidants (N+anti-ox), hypercholesterolemic (HC) and HC+anti-ox pigs

 
3.2. NF-{kappa}B and eNOS protein expression and immunoreactivity
There was a significant increase in protein expression of activated p65 subunit NF-{kappa}B in the coronary arteries from the HC animals as compared to animals on normal diet (Fig. 1, top panel). Immunohistochemical studies confirmed the presence of the activated p65 subunit of NF-{kappa}B in the arterial wall that was localized mainly in the endothelial layer (Fig. 1, bottom panel). However, in HC animals receiving chronic antioxidant supplementation, NF-{kappa}B protein expression was similar to normal animals (P=0.36), and significantly lower than in HC animals (P=0.015; Fig. 1).


Figure 1
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  		Fig. 1 Top panel: densitometric analysis of NF-{kappa}B protein expression (Western blotting) of porcine coronary tissue homogenates of normal (n=4), hypercholesterolemic (HC, n=4) and HC animals treated with antioxidants (HC+anti-ox, n=4). Results are the mean±S.E.M. Bottom panel: immunostaining of coronary epicardial vessels for NF-{kappa}B in the same pigs (p65 subunit of NF-{kappa}B, 1:25 dilution of 1 mg/ml of antibody).

 
Protein expression and localization of eNOS was similar in both groups of normal animals (Fig. 2). In contrast, in HC animals there was a decrease in eNOS protein expression in the coronary artery segments compared to controls, as assessed by immunoblotting (Fig. 2, top). Similar to NF-{kappa}B activation, this impairment was mostly evident in the endothelial layer (Fig. 2, bottom). However, when HC pigs received chronic antioxidant supplementation, coronary artery eNOS protein expression was similar to normal-diet pigs and significantly higher compared to HC pigs (Fig. 2, top). There was a significant inverse correlation between the protein expression of NF-{kappa}B and eNOS (r=–0.88, P=0.021) in this group.


Figure 2
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  		Fig. 2 Top panel: densitometric analysis of eNOS protein expression (Western blotting) of porcine coronary tissue homogenates of normal (n=4), normal animals treated with antioxidants (normal+anti-ox, n=5), hypercholesterolemic (HC, n=5) and HC+anti-ox (n=5). Results are the mean±S.E.M. Bottom panel: immunostaining of coronary epicardial vessels for eNOS in pigs from the same groups (1:400 dilution of 250 mg/ml of antibody).

 
3.3. Oxidative stress
HC animals had decreased coronary artery tissue levels of endogenous vitamin E compared to both groups of pigs receiving normal diet (Table 2). These changes were associated with a significant decrease of the endogenous activity of cellular radical scavenger enzymes (Table 2), suggesting that HC may affect different pathways in the pro-oxidant cascade. In coronary arteries of HC animals that received daily antioxidant supplementation, there was normalization of these parameters of oxidative status (Table 2). The activity of all the endogenous enzymes in this group showed inverse correlation with NF-{kappa}B activation and that of glutathione peroxidase and Mn–SOD achieved statistical significance (r=–0.96, P=0.0006 and r=–0.834, P=0.0196, respectively).


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  		Table 2 Radical scavengers and vitamin E and C levels in coronary artery tissue, in normal pigs that received daily supplementation of a combination of the antioxidants vitamin E and C (N+anti-ox), hypercholesterolemic (HC), and HC+anti-ox

 
3.4. Nitric oxide pathway (smooth muscle cell cGMP production and vascular endothelial function)
3.4.1. Smooth muscle cell cGMP production
Under basal conditions, there was no statistically significant difference in cGMP levels in coronary arteries of normal, HC, normal+anti-ox or HC+anti-ox animals (ANOVA, P=0.856). In contrast, in response to exogenous NO donation HC animals had increased smooth muscle cell cGMP production compared to normal animals (HC: 99.2±11.4 vs. N: 67.1±3.7 and N+anti-ox: 31.2±4.0 pg/mg protein, respectively, both P=0.007). However, when HC animals received chronic antioxidant supplementation there was a preservation of smooth muscle cell cGMP production (48.4±10.1 pg/mg protein), which was similar to normals (P=NS) and different from HC animals (P=0.002).

3.4.2. Vascular endothelial function
The response of the epicardial vessels to cumulative concentrations of endothelin-1 was similar among the four groups studied (maximal contraction in normal: 10.5±1 g tension; normal+anti-ox: 13.5±1.5 g tension, HC group: 10.6±1.2 g tension, HC+anti-ox: 10.4±1.8 g tension; ANOVA, P=0.124), and there was no difference in the precontraction obtained with endothelin-1 among the four groups. There was no difference in relaxation to bradykinin in the two groups that received normal diet (Fig. 3). In epicardial coronary vessels from HC pigs, the relaxation response to bradykinin was significantly attenuated when compared to animals fed a normal diet (Fig. 3). This attenuated response was normalized in coronary epicardial arteries from HC pigs that were chronically treated with antioxidant vitamins (Fig. 3). Similar results were observed when vessels from these experimental groups were exposed to increasing doses of substance P (maximal relaxation=normal: 76±5%, normal+antiox: 72±9%, HC: 51±10%, and HC+antioxidants: 73±6%, P<0.05 normal animals or HC+ antioxidants vs. HC, P=NS normal animals vs. HC+ antioxidants). There was no difference in the vasorelaxation to papaverine, an endothelium-independent agent, among the four groups.


Figure 3
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  		Fig. 3 Epicardial arterial vasorelaxation response to bradykinin in normal (n=9), normal chronically treated with antioxidants (normal+anti-ox, n=6), HC (n=8), and HC+anti-ox, n=6. * P<0.05 compared to normal and HC+anti-ox pigs.

 
There was no difference in the mean diameter of the micro vessels among the four experimental groups (normal: 414±41, normal+ anti-ox; 307±49, HC: 340±18, and HC+anti-ox: 336±23 µm). Arterioles from HC animals exposed to bradykinin had a blunted vasodilatory response compared to both groups of normal pigs (Fig. 4). Furthermore, in HC pigs chronically treated with antioxidants, the maximal vasorelaxation response to bradykinin (Fig. 4) was similar to that observed in animals fed a normal diet (P=NS), but significantly different from HC animals (P=0.009).


Figure 4
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  		Fig. 4 Microvessels vasorelaxation response to bradykinin in normal (n=9), normal chronically treated with antioxidants (normal+anti-ox, n=6), HC (n=8), and HC+ anti-ox pigs (n=6). * P<0.05 compared to normal and HC+anti-ox pigs.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This study demonstrates, for the first time, that restoration of the endogenous redox status with chronic antioxidant intervention in experimental HC attenuated NF-{kappa}B activation in the arterial wall in association with restoration of NO bioavailability and preservation of coronary endothelial function. This study supports a role for the increased endogenous oxidative stress in vivo and activation of NF-{kappa}B in early coronary atherosclerosis.

NF-{kappa}B is a transcriptional factor that has been linked to a number of diseases associated with acute and chronic inflammation and proliferation [6]. It consists mainly of two subunits, a 65-kDa protein (p65) and a 50-kDa protein (p50). In a normal state, NF-{kappa}B remains inactive in the cytoplasm and bound to the inhibitory protein I-{kappa}B. NF-{kappa}B is activated in response to certain stimuli, such as oxidized LDL [19] and TNF-{alpha} [5]. Upon stimulation, the I-{kappa}B fraction is phosphorylated and detached from the NF-{kappa}B, resulting in translocation of activated NF-{kappa}B into the nucleus, where it binds to specific gene promoters. Indeed, many of the genes regulated by NF-{kappa}B, such as VCAM and ICAM play a critical role in the atherogenic process [20,21]. The likely early involvement of NF-{kappa}B in this process is underscored by a recent observation of strong primed activation of NF-{kappa}B signal transduction pathways in mice aortic regions predisposed to atherosclerotic lesion formation. Thus, the role of NF-{kappa}B in atherogenesis may carry significant relevance.

Increased oxidative stress and redox imbalance have also been consistently implicated in the pathogenesis of atherosclerosis [1,22,23] and a growing body of evidence indicates that activation of NF-{kappa}B may be regulated by oxidation-sensitive mechanisms [5]. Indeed, in previous studies we have demonstrated the activation of NF-{kappa}B in the intima and endothelial cells [8]. However, we can not rule out that in other models of vascular injury, NF-{kappa}B activation may occur in other vascular cells such as smooth muscle cells [24]. The increased oxidative stress observed in certain pathophysiological states, such as HC, is often characterized by decreased tissue levels or activity of the endogenous antioxidant defense systems (e.g. endogenous antioxidant vitamins and radical scavenger enzymes) [25]. While a decrease in antioxidant vitamin E levels could result from consumption and breakdown of this antioxidant to {alpha}-tocopheroxyl during the process of lipid peroxidation [25–27], the precise mechanism that could account for the decrease in the endogenous scavenger enzyme activity during increased oxidative stress remains elusive [25]. Furthermore, strong support for the involvement of ROS, and many other radicals, as common activators of NF-{kappa}B is provided by the recent observation that anti-oxidants or radical scavengers attenuated ox-LDL mediated NF-{kappa}B activation in cell cultures [28,29]. While activation of NF-{kappa}B has been linked to increased oxidative stress [5,30,31], the physiological significance of this association in early atherosclerosis remained unclear. Previous studies have demonstrated that in vivo antioxidant supplementation attenuates the production of free radicals [32], but the precise mechanism underlying this effect of antioxidant therapy on the endogenous antioxidant scavenger system remained unclear. The current study demonstrates, for the first time, that in the coronary vasculature of HC animals chronic administration of combined anti-oxidants in vivo normalized the endogenous antioxidant enzyme system. It remains to be elucidated whether this is a direct effect on enzyme activity or secondary to decreased abundance of free radicals.

In addition, this study also demonstrates that antioxidant supplementation also attenuates HC-induced NF-{kappa}B activation and preserves NO bioavailability. Thus, our study suggests that a pathologic milieu, formed by an increase in oxidative stress and impaired NO bioavailability, may be involved in the activation in NF-{kappa}B seen in experimental HC. Although this does not mandate a causal relationship, our hypothesis is underscored by the strong correlation observed between parameters of altered oxidative status as well as eNOS with activation of NF-{kappa}B. Importantly, our study also demonstrates that chronic combined antioxidant intervention preserves coronary endothelial function in experimental HC. The observation that chronic antioxidant supplementation did not affect endothelial function in pigs on normal diet suggests that antioxidant intervention may be particularly effective in pathophysiological states associated with an increase in oxidative stress. The coronary vascular relaxation response to endothelium-dependent vasodilators was restored, as was cGMP production. In pathophysiological states like HC that are associated with decreased NO bioavailability, and to compensate for this impairment, the vascular wall becomes ‘sensitize’ to NO [16,33]. This is evidenced by elevated smooth muscle cell levels of cGMP, the principal second messenger of NO, in response to exogenous NO donation. This study confirms those observations and corroborates the close relationship that exists between increased oxidative stress and the NO system.

Furthermore, coronary endothelium-dependent relaxation was preserved not only in the epicardial vasculature but in the microvasculature as well. The functional significance of the preservation of the coronary endothelial function is underscored by our previous observation that antioxidant vitamin supplementation preserves myocardial perfusion responses in experimental HC [12]. We have previously demonstrated that coronary endothelial dysfunction in humans has prognostic implications in predicting adverse cardiovascular events and long term outcome [34] and antioxidant supplementation may thus potentially have clinical benefits. Specifically, vitamin E ({alpha}-tocopherol) [26] and vitamin C (ascorbic acid) [35] have been shown to have potent antioxidants properties, and their interaction may result in a synergistic enhancement of their antioxidant capacity [13,14]. Clinically, the role of antioxidant vitamins in atherosclerosis remains to be defined [36–38], possibly because the benefits of antioxidants likely depend, among the rest, on the duration, dose, timing of the intervention [36], and combination of dietary supplementation, as recently reviewed in detail [37,39].

The term endothelial dysfunction may reflect several pathological conditions, including altered anti-inflammatory properties of the endothelium, impaired modulation of vascular growth and impairment of endothelium-dependent vasorelaxation [23]. The common pathway for these alterations may be an imbalance between the decline in the endogenous anti-oxidative defense mechanisms, an increase in oxidative stress and a decrease in NO bioavailability [23,40], all of which may alter endothelium-dependent vascular relaxation. In addition to its momentous role in the regulation of vascular tone, NO is also involved in regulation of pro-atherogenic gene expression. It may be postulated that the ability of NO to inhibit the gene expression of inflammatory mediators important in atherosclerosis is at least in part mediated via a reduction in NF-{kappa}B activity and stabilization of its cytosolic inhibitor, I-{kappa}B [5,9]. Thus, activation of NF-{kappa}B may potentially have a pivotal role in the context and the pathological scenario associated with endothelial dysfunction. This hypothesis is supported by the current study which demonstrated that the restoration of the functional NO pathway with anti-oxidant intervention also attenuated the activation of NF-{kappa}B.

In summary, this study demonstrates for the first time that in vivo chronic interruption of the endogenous oxidative stress cascade reduces HC-induced NF-{kappa}B activation, and normalizes NO bioactivity, in association with preservation of coronary endothelial function. This study suggests a role for increased oxidative stress and NF-{kappa}B activation in early atherosclerosis.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by National Institutes of Health (grants HL 03621, HL 63282, and HL 63911), the Miami Heart Research Institute, the Bruce and Ruth Rappaport Program in Vascular Biology, the Mayo Foundation, and IS.NIH grant 56980/99. The authors are grateful to Paula Carlson, Drs Yang Lee and Filomena de Nigris for their technical support.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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