Modulation of expression of endothelial nitric oxide synthase by nordihydroguaiaretic acid, a phenolic antioxidant in cultured endothelial cells

Article abstract of DOI:10.1124/mol.56.1.116

Retrospective epidemiological studies have suggested that antioxidant therapy may decrease cardiovascular morbidity and mortality rates, although the mechanisms for this effect remain unclear. In the present study, we demonstrate that selective antioxidan

Full text of DOI:10.1124/mol.56.1.116

0
026-895X/99/010116-08$3.00/0
Copyright © The American Society for Pharmacology and Experimental Therapeutics
All rights of reproduction in any form reserved.
MOLECULAR PHARMACOLOGY, 56:116–123 (1999).
Modulation of Expression of Endothelial Nitric Oxide Synthase
by Nordihydroguaiaretic Acid, a Phenolic Antioxidant in
Cultured Endothelial Cells
SANTHINI RAMASAMY, GRANT R. DRUMMOND, JOON AHN, MICHAL STOREK, JAN POHL,
SAMPATH PARTHASARATHY, and DAVID G. HARRISON
Division of Cardiology (S.R., G.R.D., J.A., D.G.H.), Department of Gynecology and Obstetrics (S.P.), and Microchemical Facility (M.S., J.P.),
Emory University, Atlanta, Georgia
Received March 30, 1999; accepted April 5, 1999
This paper is available online at http://www.molpharm.org
ABSTRACT
Retrospective epidemiological studies have suggested that an- was observed with Western blot analysis after treating BAECs
tioxidant therapy may decrease cardiovascular morbidity and or human aortic endothelial cells with NDGA. Exposure of
mortality rates, although the mechanisms for this effect remain BAECs to NDGA enhanced NO production, as measured by
unclear. In the present study, we demonstrate that selective electron paramagnetic resonance spin trapping and eNOS ac-
14
14
antioxidants can enhance expression of endothelial nitric oxide tivity, as measured by [ C]arginine-to-[ C]citrulline assay.
synthase (eNOS). We found that the antioxidants nordihy- Methylation of the phenolic hydroxyl groups completely inhib-
droguaiaretic acid (NDGA), catechol, glutaryl probucol, and ited the NDGA effect on eNOS mRNA levels. This effect of
N-acetylcysteine increased eNOS expression in cultured bo- NDGA was not due to inhibition of lipoxygenase because cis-
vine aortic endothelial cells (BAECs). NDGA seemed to be the 5,8,11,14-eicosatetraynoic acid did not alter eNOS expression.
most potent of the phenolic antioxidants, producing a 3-fold We conclude that antioxidants may not only increase the bio-
increase in eNOS mRNA. This effect of NDGA was enhanced by activity of nitric oxide but also enhance expression of the eNOS
nonphenolic antioxidants such as N-acetylcysteine and ascor- enzyme. Such an effect may prove useful in conditions such as
bic acid. Nuclear run-on studies indicated that NDGA increased hypertension and atherosclerosis, in which nitric oxide produc-
eNOS transcription. A similar increase in eNOS protein content tion and/or biological activity is impaired.
Endothelium-derived nitric oxide (NO) plays an important that there is increased production of vascular superoxide,
role in the modulation of vascular tone and regulation of which on reaction with NO results in loss of biological activ-
blood pressure. Moreover, NO inhibits platelet aggregation, ity of NO.
smooth muscle proliferation, and leukocyte adhesion to the
endothelium (Moncada and Higgs, 1993; Forstermann et al., or hypertension or who are cigarette smokers, acute infu-
994). In several pathological conditions, such as hypercho- sions of ascorbic acid have been shown to improve vasorelax-
In patients with non-insulin-dependent diabetes mellitus
1
lesterolemia, atherosclerosis, diabetes mellitus, and hyper- ation (Heitzer et al., 1996; Ting et al., 1996, 1997; Solzbach et
tension, endothelium-dependent relaxation is impaired. Re- al., 1997). In cholesterol-fed rabbits, the impairment in en-
active oxygen species appear to play an important role in the
progression of most of these disease states (Freiman et al.,
dothelium-dependent relaxation is, in part, reversed by
treatment with antioxidants such as vitamin E (Keaney et
al., 1993, 1994). Even oral administration of antioxidants has
been shown to improve endothelium-dependent vasodilation
1
986; Giugliano et al., 1995; Keaney and Vita, 1995; Rajago-
palan et al., 1996). More specifically, it has been observed
(
Simon et al., 1993; Levine et al., 1996). Although the mech-
anism by which antioxidant treatment improves vasorelax-
ation is not clear, it is generally assumed that antioxidants
scavenge superoxide and thus increase the availability of
functional NO. Kinetic constants, however, suggest that the
rate of reactions between common antioxidants and superox-
This work was supported by National Institutes of Health Grants
HL390006 and HL48667m as well as American Heart Association Scientist
Development Grant Award 9630119N (to S.R.). Portions of this work were
presented at the American Heart Association 70th Meeting, November 1997,
Orlando, FL; Society of Toxicology 37th Meeting, Seattle, WA, March 1998;
and Vascular Biology, 1998, San Francisco, CA, April 1998.
ABBREVIATIONS: NO, nitric oxide; NDGA, nordihydroguaiaretic acid; BHT, butylated hydroxy toluene; M199, Medium 199; EPR, electron
paramagnetic resonance; MGD, N-methyl-D-glucamine dithiocarbamate; eNOS, endothelial nitric oxide synthase; HAEC, human aortic endothelial
G
cell; NAC, N-acetylcysteine; L-NAME, N -nitro-L-arginine methyl ester; BAEC, bovine aortic endothelial cell; SOD, superoxide dismutase; Sp1,
simian virus 40 promoter factor 1; AP-1, activator protein 1; EGM, endothelial cell growth media; ARE, antioxidant response element; ETYA,
cis-5,8,11,14-eicosatetraynoic acid.
116

Antioxidant and Endothelial NOS Gene Regulation
117
ide is much slower than the rate of the reaction between
superoxide and NO (Forman and Fridovich, 1973; Afenas’ev,
Materials and Methods
Cell Culture and Experimental Media Preparation. Bovine
aortic endothelial cells (BAECs) were cultured in Medium 199
M199; GIBCO, Grand Island, NY) containing 10% FCS (Hyclone
Laboratories, Logan, UT) as described previously (Mugge et al.,
991). Postconfluent cells between passages 5 and 10 were used for
1
991; Huie and Padmaja, 1993). This raises the possibility
that antioxidants may have other effects on the endothelial
arginine/NO pathway beyond scavenging superoxide and
prolonging the half-life of NO.
(
1
Recent evidence suggests that antioxidants can modulate
gene expression. For example, antioxidant treatment has
been shown to induce expression of chemoprotective enzymes
like glutathione S-transferase (Rushmore et al., 1991) and
NAD(P)H:quinone reductase (Favreau and Pickett, 1993)
and thus prevent cells from malignant transformation. In
addition, phenolic antioxidants have been shown to inhibit
expression of genes involved in inflammatory processes such
the experiments. Human aortic endothelial cells (HAECs) were ob-
tained from Clonetics (Walkersville, MD) and cultured in endothelial
cell growth media (EGM; Clonetics) containing 2% FCS.
Postconfluent endothelial cells were exposed to control (M199 plus
5% FCS in the case of BAECs or EGM plus 2% FCS in the case of
HAECs) or media supplemented with one of several phenolic anti-
oxidants, including nordihydroguaiaretic acid (NDGA), vitamin E,
catechol, caffeic acid, butylated hydroxy toluene (BHT), and glutaryl
as vascular cell adhesion molecule and intercellular adhesion probucol, a water-soluble form of probucol (Table 1). In other exper-
molecule (Cominacini et al., 1996). Of interest, the promoter iments, water-soluble antioxidants such as ascorbic acid, cysteine, or
of human endothelial nitric oxide synthase (eNOS) gene con- N-acetylcysteine (NAC) were used. Concentrated stocks of phenolic
tains simian virus 40 promoter factor 1 (Sp-1), activator antioxidants were made in ethanol, and the appropriate aliquots
protein 1 (AP-1), and antioxidant response elements, all of were added to M199 plus 5% FCS in the case of BAECs or EGM in
which are regulated by cellular redox state (Sen and Packer, the case of HAECs to prepare antioxidant-enriched media. The con-
centration of ethanol in the antioxidant-enriched media was never
1
996; Palmer and Paulson, 1997). Based on these consider-
higher than 0.1%. In the case of water-soluble antioxidants, concen-
trated stocks were made in distilled water and sterile filtered, and
appropriate aliquots were added to M199 plus 5% FCS or EGM.
ations and the previous findings in experimental animals, we
performed the present study to test the hypothesis that an-
tioxidants might modulate eNOS gene expression.
TABLE 1
Effect of various phenolic antioxidants on eNOS mRNA levels
Name and Structure of Antioxidant
eNOS/28s
␮
M
fold increase above control
NDGA
16
1.8 Ϯ 0.1
2.9 Ϯ 0.2
3
2
Catechol
50
00
1.4 Ϯ 0.04
3.2 Ϯ 0.2
1
Caffeic acid
100
0.8 Ϯ 0.04
0.9 Ϯ 0.03
2
00
Vitamin E
BHT
25
1.0 Ϯ 0.02
1.0 Ϯ 0.1
5
0
50
00
0.8 Ϯ 0.03
Toxic
1
Glutaryl probucol
10
1.7 Ϯ 0.1
Toxic
2
5

1
18
Ramasamy et al.
Synthesis of Methylated NDGA. NDGA (100 mg; 0.33 mmol) macia Biotech, Piscataway, NJ). Membranes were hybridized for 60
was dissolved in 0.21 ml (3.3 mmol) of iodomethane and 5 ml of dry min at 68°C using Quick Hyb (Stratagene) and washed subsequently
acetone. Potassium carbonate (0.92 g) was then added to the mix- according to the conditions described previously (Ramasamy et al.,
ture, and the mixture was refluxed for approximately 10 h. Complete 1998). The blots were visualized using a PhosphorImager. Intensity
methylation was monitored by thin-layer chromatography on silica of the eNOS bands was quantified using ImageQuant software (Mo-
plates using ethyl acetate/petrol ether (19:1, v/v) as the mobile phase, lecular Dynamics, Sunnyvale, CA). The intensities of the eNOS
and the detection was done using iodine vapors. The reaction mix- mRNA bands were normalized for the corresponding 28S band or
ture was evaporated on a rotary evaporator and extracted with ether. 18S bands.
After evaporation of ether, the residue was dissolved in 2.0 ml of
methanol/water (17:3, v/v).
NOS Activity Assay. Postconfluent BAECs were treated with
control or antioxidant-containing media for 24 h. Whole-cell homog-
Methylated NDGA (Me NDGA) was purified using reversed phase enates (ϳ250 ␮g total protein) were then used for NOS activity assay
4
HPLC on a C18 Microsorb-MV silica column (5 ϫ 250 mm, dp ϳ5 ␮m, as previously described, and the converted citrulline was separated
ϩ
1
00-A pore size; Rainin Instruments) at a flow rate of 0.5 ml/min at from labeled L-arginine using a Dowex AG 50WX-8 (Na form; Bio-
room temperature. The column was developed using methanol/water Rad, Hercules, CA) (Ramasamy et al., 1998). Radioactivity in the
4:1, v/v) under isocratic conditions, followed by UV detection at 280 eluate was determined using LS6500 Beckman Liquid Scintillation
(
nm. The retention time of completely methylated NDGA was 29 min. Counter (Beckman Instruments, Inc., Schaumburg, IL). Protein con-
The mass of Me NDGA was confirmed by matrix-assisted laser centration in the homogenate was determined using Bio-Rad DC
4
desorption/ionization time-of-flight mass spectrometry, performed reagent.
using the Bruker ProteinTOF instrument. The sample was prepared
EPR Spin-trapping for Measurement of NO Production.
according to Storek and Pohl (1997). Briefly, 1.5 ␮l (1 nmol) of the Confluent BAECs grown in 6-well plates were treated with M199
sample was mixed with 5 ␮l of the saturated solution of indole-2- with 5% FCS containing 0 or 25 ␮M NDGA for 24 h. The cells were
carboxylic acid. An aliquot (0.5 ␮l) of this mixture was deposited on washed in Krebs/HEPES buffer containing 99.01 mM NaCl, 4.69 mM
the target, air dried, and analyzed. Spectra were obtained by aver- KCl, 1.87 mM CaCl2 1.2 mM MgSO , 1.03 mM K HPO , 25.0 mM
4
2
4
aging 50 single-laser-shot spectra. Parameters were set for laser NaHCO , 20.0 mM Na-HEPES, and 11.1 mM glucose, pH 7.4, and
3
wavelength of 337 nm, ion acceleration voltage of 17.5 kV, and incubated subsequently with 1.5 ml of the same buffer containing 1.5
reflector voltage of 20 kV.
mM N-methyl-D-glucamine dithiocarbamate (MGD; Oxis Interna-
Northern Blotting. Total RNA was extracted using TRI reagent tional, Inc., Portland, OR), 0.3 mM ferrous sulfate, and 100 U/ml
2ϩ
2ϩ
(
Molecular Research Center, Inc., Cincinnati, OH). RNA (20 ␮g) was Cu /Zn superoxide dismutase (SOD) at 37°C. For NO stimula-
loaded on a 1.0% agarose/3% formaldehyde gel, electrophoresed, and tion, cells were incubated in the same buffer mixture containing
G
transferred to a nitrocellulose membrane overnight. A full-length either 1 ␮M A23187 or bradykinin. In some experiments, N -nitro-
(
[
4.2 kb) radioactive-labeled eNOS probe was prepared using
L-arginine methyl ester (L-NAME, 1 mM) was added 30 min before
P]dCTP (Dupont NEN, Boston, MA) and oligolabeling kit (Phar- agonist stimulation. After a 30-min incubation, the Krebs/HEPES
buffer was removed and immediately frozen in liquid nitrogen. The
32
cell monolayers were dissolved in 0.5% SDS, and total cellular pro-
tein content was determined as mentioned above. EPR spectra of the
2ϩ
MGD-Fe -NO complex were recorded using a Bruker EMX EPR
spectrometer (Bruker Instruments Inc., Billerica, MA) at microwave
frequency of 9.785 GHz, microwave power of 20 mW, modulation
frequency of 100 kHz, and modulation amplitude of 2.5 G, at room
temperature. A standard curve was obtained by incubating the
2ϩ
MGD-Fe complex with 0 to 5 ␮M concentration of the NO donor
-(N,N-diethylamino)diazenolate-2-oxide, sodium salt (Alexis Bio-
2
chemicals, San Diego, CA) for 30 min at 37°C, and EPR spectra were
obtained under the same conditions as mentioned above for samples.
Peak intensity of the EPR spectra was proportional to the amount of
Fig. 1. Northern blot analysis showing the effect of increasing concen-
trations of NDGA on eNOS mRNA in BAECs. Postconfluent BAECs were
exposed to 0 to 32 ␮M NDGA in M199 plus 5% FBS for 24 h. A, repre-
sentative blot. B, combined densitometric ratio of eNOS/28s RNA. C,
effect of concomitant addition of either Cu /Zn SOD or Ca -EDTA
with NDGA on eNOS expression. BAECs were exposed to either 16 ␮M
NDGA or SOD (100 U/ml) or 100 ␮M EDTA individually or in combina-
tion for 24 h. D, percent eNOS/28s rRNA ratio from three separate
experiments similar to those shown in C. Data are presented as mean Ϯ
S.E.M.
Fig. 2. Nuclear run-off analysis demonstrating the effect of 16 ␮M NDGA
on the transcription rate of the eNOS gene in BAECs. Nuclei were
harvested from endothelial cells exposed to control and NDGA-containing
2ϩ
2ϩ
2ϩ
32
media for 4 h. Nascent transcripts were P-labeled, and equal counts
from each group were added to the blots containing eNOS, ␤-actin, and
vector DNA. Hybridizations were performed for 48 to 72 h at 65°C. The
blots were washed and exposed to PhosphorImager. A, representative
blots. B, densitometric ratios of eNOS/␤-actin bands. Data are presented
as mean Ϯ S.E.M. for three separate experiments.

Antioxidant and Endothelial NOS Gene Regulation
119
2ϩ
MGD-Fe -NO complex in the Krebs/HEPES buffer. The total peak visualized using a PhosphorImager and quantified using Image-
intensities of the samples were compared with that obtained for Quant software.
standards allowing quantification of the amount of NO produced by
the cells. NO production was normalized to total cellular protein
content.
Results
Western Blotting. BAECs or HAECs were harvested, and the
homogenates were prepared as described in NOS assay. Then, 20 ␮g
of protein was electrophoresed using 7.5% SDS-polyacrylamide gel
and transferred to a nitrocellulose membrane. Western analysis was
performed using a 1:2000 dilution of a monoclonal antibody against
human eNOS (Transduction Laboratories, Lexington, KY) and a
secondary goat anti-mouse antibody conjugated to horseradish per-
oxidase (Amersham Corporation, Arlington Heights, IL) (Ramasamy
et al., 1998). Signals of the immunoreactive bands were measured
using the ECL detection system (Amersham).
NDGA Treatment Causes Dose-Dependent Increase
in eNOS mRNA Levels. Exposure of BAECs to increasing
concentrations of NDGA (0–32 ␮M) for 24 h caused a dose-
dependent increase in eNOS mRNA levels as measured by
Northern analysis (Fig. 1A). Densitometric readings of the
Northern blots indicated that there was an 80 and a 300%
increase in the ratios of eNOS to 28S band after 16 and 32
␮M NDGA treatments, respectively (Fig. 1B).
Nuclear Run-On Assay. Nuclear run-on assays were performed
Phenolic compounds like NDGA may have both antioxi-
using a method described by Greenberg (1987) with modifications dant and prooxidant effects. In the latter case, the phenolic
(
1
Ramasamy et al., 1998). Briefly, BAECs were treated with control or
6 ␮M NDGA-containing media for 4 h. Cells were harvested and
group is often oxidized to a phenoxyl radical, which then can
exert prooxidant effects (Santanam and Parthasarathy,
lysed in 0.5% Nonidet P-40, and the nuclei were isolated by centrif-
ugation. In vitro transcription was performed using 5 ϫ 10 nuclei in
a reaction buffer containing a 2 mM concentration each of ATP, GTP,
1995). Common mechanisms involve oxidation catalyzed by
7
transition metals and reactions with superoxide. To examine
the possibility that NDGA exerted its effect on eNOS mRNA
via such a mechanism, cells were concomitantly treated with
32
and CTP and 100 ␮Ci of [␣- P]UTP (DuPont-NEN) for 30 min at
0°C. Total RNA was extracted, and the radiolabeled transcripts
3
(
2ϩ
2ϩ
6
NDGA plus either SOD or Ca -EDTA. Neither Ca -EDTA
total activity, approximately 5 ϫ 10 cpm) were hybridized to nylon
membranes previously slot-blotted with eNOS cDNA, ␤-actin cDNA, nor SOD affected eNOS mRNA levels alone, but both aug-
and the full-length pBluescript vector. Bands were subsequently mented the effect of NDGA (Fig. 1, C and D). These data
Fig. 3. Northern blot analysis exam-
ining the effect of nonphenolic antioxi-
dants on NDGA-induced eNOS ex-
pression. A, Northern blot from
BAECs treated for 24 h with 100 ␮M
ascorbic acid or 200 ␮M cysteine, ei-
ther alone or in combination with 16
␮M NDGA. B, percent densitometric
ratio of eNOS/28s rRNA from ascorbic
or cysteine groups. C, Northern blot
from BAECs treated with 0.5 or 5.0
mM NAC either alone or in combina-
tion with 16 ␮M NDGA for 24 h. D,
percent densitometric ratio of eNOS/
2
8s rRNA from NAC groups. Data are
presented as mean Ϯ S.E.M. for three
separate experiments.

1
20
Ramasamy et al.
suggest that the effect of NDGA on eNOS mRNA levels and ascorbic acid or cysteine caused a 2.4- and 1.9-fold in-
involves an antioxidant rather than a prooxidant effect.
The structures of several antioxidants, including NDGA,
crease in eNOS mRNA levels, respectively (Fig. 3B).
Similarly, another nonphenolic antioxidant, NAC, when
and their effects on eNOS mRNA levels are shown in Table 1. used alone, had no effect on eNOS mRNA levels when added
In some instances, the use of higher concentrations of some in low concentrations (0.5 mM) for 24 h. A higher concentra-
phenolic antioxidants (BHT and probucol) was limited due to tion of NAC (5 mM) produced a 90% increase in eNOS mRNA,
cellular toxicity.
similar to that observed on exposure of cells to 16 ␮M NDGA.
NDGA-Induced Increase in eNOS mRNA levels Is Interestingly, an additive increase in eNOS mRNA levels
Transcriptionally Mediated. NDGA treatment caused an was found when cells were treated with NDGA and 5.0 mM
increase in the transcription rate of the eNOS gene but not NAC (Fig. 3, C and D).
for ␤-actin (Fig. 2A). The hybridization of nascent transcripts
NDGA and NAC Treatments Increase eNOS Protein
to eNOS as well as ␤-actin cDNA in the blots was specific as and Enzyme Activity. Because NDGA and NAC exerted
the hybridization to vector DNA was negligible. Quantitative the most impressive effects on eNOS mRNA levels, studies
results from three separate experiments indicated that the examining eNOS protein expression and enzyme activity
ratio of eNOS to ␤-actin transcription increased 2-fold as a were focused on the effects of these compounds. When used
result of NDGA treatment (Fig. 2B).
alone, NDGA (16 ␮M) and NAC (5 mM) increased the eNOS
Nonphenolic Antioxidants Augment NDGA-Induced protein content 2- and 2.3-fold, respectively, compared with
Increase in eNOS mRNA Levels. In BAECs, relatively low control BAECs. The combined exposure of NDGA and NAC
concentrations of ascorbic acid (100 ␮M) or cysteine (200 caused an additive increase (3.5-fold) in eNOS protein con-
␮M), when given alone, failed to alter eNOS mRNA levels. In tent (Fig. 4, A and B). A qualitatively similar effect in eNOS
contrast, 100 ␮M ascorbic acid substantially enhanced the protein expression was observed in HAECs treated with NDGA,
effect of 16 ␮M NDGA on eNOS expression. Quantitative NAC, and the combination of the two (Fig. 4, C and D).
densitometry indicated that combined treatment with NDGA
In correspondence with the increase in eNOS protein con-
tent, both NDGA (16 ␮M) and NAC (5 mM) elevated eNOS
enzyme activity by approximately 2-fold (Fig. 5). Interest-
ingly, the combination of NDGA and NAC, which had a
dramatic effect on eNOS protein content, did not increase
eNOS enzyme activity beyond that observed when either
agent was used alone (Fig. 5).
Effect of NDGA on Endothelial Cell NO Production.
NDGA (25 ␮M) elevated NO production as estimated by EPR
(
Fig. 6) in unstimulated cells as well as cells treated with
either A23187 or bradykinin. Representative spectra of
2ϩ
MGD-Fe -NO complexes obtained from control and NDGA-
treated BAECs under basal and agonist-stimulated condi-
tions are depicted in Fig. 6A. Analysis of mean data indicated
that NDGA approximately doubled NO production under
Fig. 4. Western blot analysis demonstrating the effect of antioxidants on
eNOS protein content in BAECs (A and B) and HAECs (C and D). Cells
were exposed to control media or media containing either 16 ␮M NDGA,
5
mM NAC, or the combination of NAC with NDGA for 24 h. Each lane
1
4
was loaded with 15 ␮g of protein. A monoclonal antibody against human
eNOS was used to detect the bands. Representative blots are illustrated
in A and C from BAECs and HAECs, respectively. The densitometric
readings as percent control of Western blots derived from BAECs and
HAECs are presented in B and D, respectively. Data are presented as
mean Ϯ S.E.M. for three separate experiments.
Fig. 5. NOS activity as measured by the conversion of [ C]arginine to
14
[
C]citrulline. Cells were exposed to control media or media containing
either 16 ␮M NDGA, 5 mM NAC, or the combination of NAC and NDGA
for 24 h. The cells were homogenized, and 250 ␮g of protein was used for
the assay. Results represent the mean Ϯ S.E.M. from three separate
experiments.

Antioxidant and Endothelial NOS Gene Regulation
121
basal conditions and in response to A23187 and increased ished in a variety of pathophysiological conditions, resulting
responses to bradykinin by 50%. NO production was virtually in impaired endothelium-dependent vasodilation. In hyper-
eliminated by 1 mM L-NAME treatment (Fig. 6B).
cholesterolemia, treatment with SOD, dietary antioxidants
NDGA-Induced Increase in eNOS Expression Is Not including beta carotene or vitamin E (Keaney et al., 1993), or
Related to Lipoxygenase Inhibition. Unlike NDGA, the the phenolic antioxidant probucol (Keaney et al., 1993) have
commonly used lipoxygenase inhibitor cis-5,8,11,14-eicosa- been shown to improve endothelium-dependent vasodilation.
tetraynoic acid (ETYA) (50 ␮M) had no effect on either eNOS Recently, antioxidant treatment has also been shown to in-
enzyme activity (Fig. 7A) or mRNA levels (7B) on 24 h treat-
ment.
Phenolic Hydroxyl Groups Are Critical for NDGA-
Induced Increase in eNOS Expression. The MALDI-TOF
mass spectrum of NDGA after O-methylation confirmed that
the molecular weight of the modified molecule, 358.4, amu
crease endothelium-dependent vasodilation and reduce blood
pressure in angiotensin II-induced hypertension (Laursen et
al., 1997). In general, it is thought that antioxidants improve
endothelium-dependent vasodilation by scavenging superox-
ide and thus increasing the availability of functional NO
(
Harrison and Ohara, 1995). The rate constants for the reac-
corresponded to the predicted molecular weight of Me NDGA
4
tion between superoxide and antioxidant vitamins such as
vitamin E and ascorbic acid, however, are approximately
(
Fig. 8A). In addition, fast atom bombardment mass spec-
trometry analysis (not shown) confirmed the presence of four
10,000 times slower than the rate constant for the reaction
methyl groups. Unlike native NDGA, Me NDGA had no ef-
4
between NO and superoxide (Forman and Fridovich, 1973;
Afenas’ev, 1991; Huie and Padmaja, 1993). Our current
study provides an additional mechanism whereby antioxi-
dants may improve endothelial-dependent vasodilation (i.e.,
by increasing eNOS expression).
Of interest, the effect of the various phenolic antioxidants
on eNOS expression varied substantially (Table 1). The an-
tioxidant properties and the propensity of these compounds
to undergo redox cycling are affected by their respective
R-groups attached to the aromatic ring. Among the phenolic
antioxidants used in this study, NDGA is the most potent. At
a concentration of 32 ␮M, NDGA increased eNOS expression
by 3-fold. NDGA has well established antioxidant properties
fect on eNOS mRNA levels (Fig. 8, B and C). These results
indicated that the 4-hydroxyl groups were critical in mediat-
ing the effect of NDGA on eNOS mRNA expression.
Discussion
The new finding of this study is that antioxidants, includ-
ing NDGA, NAC, probucol, and catechol, enhance the expres-
sion of eNOS in cultured endothelial cells. The most potent of
these, the phenolic antioxidant NDGA, increased eNOS ex-
pression by as much as 3-fold. This effect of NDGA is at least
in part transcriptionally mediated and is augmented by the
presence of nonphenolic small antioxidant molecules such as
ascorbic acid and NAC. In addition, increased eNOS gene and is known to prevent oxidation of lard stored at room
expression by NDGA in endothelial cells is mediated by its temperature for up to 1 year (Lundberg et al., 1944). NDGA
phenolic groups, as the methylation of these groups abolished has previously been shown to be more potent than vitamin E,
this effect of the compound. Finally, NDGA-mediated in- probucol, caffeic acid, or BHT in preventing oxidation of
creases in eNOS gene expression are likely due to antioxi- linoleic acid (Sgargali et al., 1993). It is also possible that
dant rather than prooxidant effects and are independent of structural characteristics and hydrophobic properties of the
lipoxygenase inhibition.
various compounds influence their capacity to interact with
The biological activity of endothelium-derived NO is dimin- redox sensitive transcription factors.
2
ϩ
Fig. 6. A, EPR spectra of MGD-Fe -NO complex obtained in control and 25 ␮M NDGA-treated BAECs under basal and agonist-stimulated conditions.
EPR spectra were obtained after incubation of the cells in Krebs/HEPES buffer containing 1.5 mM MGD, 0.3 mM ferrous sulfate, and 100 U/ml
2ϩ
2ϩ
Cu /Zn SOD at 37°C for 30 min. The cells were stimulated either with 1 ␮M A23187 or bradykinin. In some cells, 1 mM L-NAME was added 30
4
min before agonist stimulation. Spectrometer settings were microwave frequency of 9.785 GHz, microwave power of 20 mW, receiver gain of 1 ϫ 10 ,
modulation frequency of 100 kHz, modulation amplitude of 2.5 G, and scan time of 83.886 s. Images are the averages of five scans subjected to baseline
correction. B, quantification of spectra obtained from samples (n ϭ 3) after correction for cellular protein. A standard curve generated by exposure of
2ϩ
MGD-Fe to 2-(N,N-diethylamino)diazenolate-2-oxide (0–5 ␮M) was used to quantify the actual amount of NO produced by the cells.

1
22
Ramasamy et al.
Our studies indicate that the phenolic hydroxyl groups of eNOS promoter. Phenolic antioxidants have been shown to
NDGA are necessary for enhancing eNOS expression. When induce expression of c-Fos, and c-Jun, resulting in enhanced
the phenolic hydroxyl groups were methylated, the ability of AP-1 binding activity in hepatocytes (Choi and Moore, 1993).
the modified compound to affect eNOS expression was com- In contrast, phenolic antioxidants may inhibit AP-1 activity
pletely abolished (Fig. 8). Thus, it seems that the presence of by increasing Fra (Yoshioka et al., 1995). It is also possible
the phenolic hydroxyl groups, which are critical for the anti- that NDGA increases eNOS expression through activation of
oxidant properties of the compound, are also important in protein or proteins binding to an antioxidant response ele-
enhancing eNOS expression.
ment (ARE). Both the human and bovine eNOS promoters
Phenolic hydroxyl-containing compounds may exert both contain sequences resembling the ARE in the NAD(P)H:
antioxidant and prooxidant properties. When a radical ab- quinone reductase and the glutathione S-transferase promot-
stracts the hydrogen atom of the phenolic group, the phenolic ers. The presence and significance of ARE-binding proteins
antioxidant is converted to a phenoxyl radical. Reducing in endothelial cells have yet to be examined.
agents such as ascorbic acid or thiol containing compounds
Interestingly, the combination of NDGA with NAC, al-
promote reduction of the phenoxyl radicals back to the parent though increasing eNOS mRNA and protein levels, did not
compound (Thomas et al., 1995). If the effect of NDGA on increase eNOS enzyme activity as determined by L-arginine-
eNOS expression were mediated via a prooxidant effect, it to-L-citrulline activity (Figs. 3 and 4 versus Fig. 5). The
would be expected that addition of reducing agents such as reason for this discrepancy is unclear, but it may be related
ascorbate, cysteine, or NAC would have diminished induction
of eNOS by NDGA. This was not found to be the case. Ascor-
bate and NAC enhanced the effect of NDGA on eNOS expres-
sion, whereas cysteine was without effect. Furthermore, the
2ϩ
2ϩ
է
addition of Cu /Zn SOD (to scavenge O2 in the media) or
2ϩ
Ca -EDTA (to chelate transition metals) enhanced the ef-
fect of NDGA. Taken together, these lines of evidence suggest
that the effect of NDGA on eNOS expression is mediated via
antioxidant rather than prooxidant properties (Fig. 1).
NDGA is also known to be nonselective lipoxygenase in-
hibitor. It is unlikely, however, that its effect on eNOS ex-
pression was mediated via lipoxygenase inhibition (Fig. 7).
Neither lipoxygenase protein nor enzyme activity has been
detected in endothelial cells. Furthermore, another potent
inhibitor of lipoxygenase, ETYA (in concentrations exceeding
that necessary to inhibit lipoxygenase), had no effect on
eNOS expression or enzyme activity (Reddy et al., 1994).
Nuclear run-on analysis suggested that NDGA enhances
the rate of eNOS mRNA transcription (Fig. 2). The promoter
of eNOS contains cis-elements that in the case of other genes,
are modulated by cellular redox state. In particular, Sp-1
binding is crucial for basal transcription of eNOS. In other
genes and tissues, antioxidant treatment has been shown to
increase Sp-1 binding, whereas the addition of hydrogen
peroxide or diamide or the depletion of glutathione decreases
Sp-1 binding. AP-1-like elements are also present in the
Fig. 8. Role of the phenolic hydroxyl groups in modulation of eNOS
expression by NDGA. After methylation of NDGA, mass spectrometric
analysis indicated a single peak at the molecular weight of 358, corre-
Fig. 7. Effect of the lipoxygenase inhibitor ETYA on eNOS expression. A,
eNOS activity as measured by the conversion of radioactive arginine to
citrulline in BAECs treated with either 16 ␮M NDGA or 50 ␮M ETYA.
Data are presented as mean Ϯ S.E.M. of three separate experiments. B,
Northern blot indicating the effect of 24-h treatment of ETYA (0–50 ␮M)
on eNOS steady-state mRNA. The experiments were repeated three
times.
sponding to the predicted molecular weight of Me NDGA (A). BAECs
4
were treated with either NDGA or Me NDGA for 24 h, and eNOS mRNA
4
levels were measured. B, representative Northern blot. C, the percent
eNOS/28s rRNA ratio from three separate experiments. Data are pre-
sented as mean Ϯ S.E.M. from three separate experiments.

Antioxidant and Endothelial NOS Gene Regulation
123
Keaney JFJr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo
J and Vita JA (1994) Low-dose ␣-tocopherol improves and high-dose ␣-tocopherol
worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest
93:844–851.
Keaney JF Jr and Vita JA (1995) Atherosclerosis, oxidative stress, and antioxidant
protection in endothelium-derived relaxing factor action. Prog Cardiovasc Dis
38:129–154.
Khan BV, Harrison DG, Olbrych MT, Alexander RW and Medford RM (1996) Nitric
oxide regulates vascular cell adhesion molecule 1 gene expression and redox-
sensitive transcriptional events in human vascular endothelial cells. Proc Natl
Acad Sci USA 93:9114–9119.
Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA and Harrison DG (1997)
Role of superoxide in angiotensin II-induced but not catecholamine-induced hy-
pertension. Circulation 95:588–593.
to either subcellular localization of the enzyme or autoinhi-
bition of enzyme activity as recently described by Griscavage
et al. (1995).
In addition to mediating endothelium-dependent vasodila-
tion, endothelium-derived NO has been shown to inhibit
platelet aggregation (Alheid et al., 1987), vascular cell adhe-
sion molecule-1 expression (Khan et al., 1996), and smooth
muscle growth (Garg and Hassid, 1989), all of which are
considered antiatherogenic properties. There is substantial
evidence that antioxidants may inhibit the atherosclerotic
process, likely via a variety of mechanisms, including pre-
vention of low-density lipoprotein oxidation and inhibition of
vascular adhesion molecule expression. Increased expression
of NO synthase and production of endothelial NO may be
another mechanism whereby antioxidants inhibit the devel-
opment of atherosclerosis. Modulation of eNOS expression by
compounds similar to NDGA may be a useful clinical target
for future drug design and clinical studies.
Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF and Vita JA (1996)
Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coro-
nary artery disease. Circulation 93:1107–1113.
Lundberg WO, Halvorson HO and Burr GO (1944) The antioxidant properties of
nordihydroguaiaretic acid. Oil Soap (Chicago) 21:33–35.
Moncada S and Higgs A (1993) The L-arginine-nitric oxide pathway. N Engl J Med
329:2002–2012.
Mugge A, Peterson T and Harrison DG (1991) Release of nitrogen oxides from
cultured bovine aortic endothelial cells is not impaired by calcium channel antag-
onists. Circulation 83:1404–1409.
Palmer HJ and Paulson KE (1997) Reactive oxygen species and antioxidants in
signal transduction and gene expression. Nutr Rev 55:353–361.
Rajagopalan S, Kurz S, M u¨ nzel T, Tarpey M, Freeman B, Griendling K and Harrison
D (1996) Angiotensin II-mediated hypertension in the rat increases vascular
superoxide production via membrane NADH/NADPH oxidase activation: Contri-
bution to alterations of vasomotor tone. J Clin Invest 97:1916–1923.
Ramasamy S, Parthasarathy S and Harrison DG (1998) Regulation of endothelial
nitric oxide synthase gene expression by oxidized linoleic acid. J Lipid Res 39:
268–276.
References
Afenas’ev I (1991) Superoxide Ion: Chemistry and Biological Implications. CRC
Press, Boca Raton, FL.
Alheid U, Fr o¨ lich JC and F o¨ rstermann U (1987) Endothelium-derived relaxing factor
from cultured human endothelial cells inhibits aggregation of human platelets.
Thromb Res 47:561–571.
Choi H-S and Moore DD (1993) Induction of c-fos and c-jun gene expression by
phenolic antioxidants. Mol Endocrinol 7:1596–1602.
Reddy RG, Yoshimoto T, Yamamoto S, Funk CD and Marnett LJ (1994) Expression
of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its
characterization. Arch Biochem Biophys 312:219–226.
Rushmore TH, Morton MR and Pickett CB (1991) The antioxidant responsive ele-
ment: Activation by oxidative stress and identification of the DNA consensus
sequence required for functional activity. J Biol Chem 266:11632–11639.
Santanam N and Parthasarathy S (1995) Paradoxical actions of antioxidants in the
oxidation of low density lipoprotein by peroxidases. J Clin Invest 95:2594–2600.
Sen CK and Packer L (1996) Antioxidant and redox regulation of gene transcription.
FASEB J 10:709–720.
Cominacini L, Garbin U, Pasini AF, Davoll A, Campagnola M, Contessi GB, Pas-
torino AM and Cascio VL (1996) Antioxidants inhibit the expression of intercellu-
lar adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized
LDL on human umbilical vein endothelial cells. Free Radic Biol Med 22:117–127.
Favreau LV and Pickett CB (1993) Transcriptional regulation of the rat NAD(P)H:
quinone reductase gene: Characterization of a DNA-protein interaction at the
antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13-
acetate. J Biol Chem 268:19875–19881.
Forman HJ and Fridovich I (1973) Superoxide dismutase: A comparison of rate
constants. Arch Biochem Biophys 158:396–400.
Forstermann U, Closs EI, Pollock JS, Nakane M, Schwarz P, Gath I and Kleinert H
Sgargali GP, Valoti M, Gorelli B, Fusi F, Palmi M and Mantovani P (1993) Calcium
antagonist and antioxidant properties of some hindered phenols. Br J Pharmacol
110:369–377.
(
1994) Nitric oxide synthase isozymes. Characterization, purification, molecular
Simon BC, Haudenschild CC and Cohen RA (1993) Preservation of endothelium-
dependent relaxation in atherosclerotic rabbit aorta by probucol. J Cardiovasc
Pharmacol 21:893–901.
Solzbach U, Hornig B, Jeserich M and Just H (1997) Vitamin C improves endothelial
dysfunction of epicardial coronary arteries in hypertensive patients. Circulation
cloning, and functions. Hypertension 23:1121–1131.
Freiman PC, Mitchell GC, Heistad DD, Armstrong ML and Harrison DG (1986)
Atherosclerosis impairs endothelium-dependent vascular relaxation to acetylcho-
line and thrombin in primates. Circ Res 58:783–789.
Garg UC and Hassid A (1989) Nitric oxide-generating vasodilators and 8-bromo-
cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured
rat vascular smooth muscle cells. J Clin Invest 83:1774–1777.
96:1513–1519.
Storek M and Pohl J (1997) Indol-2-carboxylic acid: A new matrix for MALDITOF
mass spectrometry. Czech Chemical Society Meeting, Zlin. Abstract.
Giugliano D, Ceriello A and Paolisso G (1995) Diabetes mellitus, hypertension, and
cardiovascular disease: Which role for oxidative stress? Metabolism 44:363–368
Greenberg M (1987) Identification of newly transcribed RNA, in Current Protocols in
Molecular Biology (Ausubel F, Brent R, Kingston R, Moore D, Smith J, Seidman J
and Struhl K eds) pp 4.10.1–4.10.11, John Wiley and Sons, New York.
Griscavage JM, Hobbs AJ and Ignarro LJ (1995) Negative modulation of nitric oxide
synthase by nitric oxide and nitroso compounds. Adv Pharmacol 34:215–234.
Harrison DG and Ohara Y (1995) Physiologic consequences of increased vascular
oxidant stresses in hypercholesterolemia and atherosclerosis: Implications for
impaired vasomotion. Am J Cardiol 75:75B–81B.
Heitzer T, Just H and Munzel T (1996) Antioxidant vitamin C improves endothelial
dysfunction in chronic smokers. Circulation 94:6–9.
Huie RE and Padmaja S (1993) The reaction of NO with superoxide. Free Radic Res
Commun 18:195–199.
Thomas SR, Neuzil J, Mohr D and Stocker R (1995) Coantioxidants make alpha-
tocopherol an efficient antioxidant for low density lipoprotein. Am J Clin Nutr
62:1357S–1364S.
Ting HH, Timimi FK, Boles KS, Creager SJ, Ganz P and Creager MA (1996) Vitamin
C improves endothelium-dependent vasodilation in patients with non-insulin-
dependent diabetes mellitus. J Clin Invest 97:22–28.
Ting HH, Timimi FK, Haley EA, Roddy M-A, Ganz P and Creager MA (1997)
Vitamin C improves endothelium-dependent vasodilation in forearm resistance
vessels of humans with hypercholesterolemia. Circulation 95:2617–2622.
Yoshioka K, Deng T, Cavigelli M and Karin M (1995) Antitumor promotion by
phenolic antioxidants: Inhibition of AP-1 activity through induction of Fra expres-
sion. Proc Natl Acad Sci USA 92:4972–4976.
Keaney JF Jr, Gaziano JM, Xu A, Frei B, Curran-Cellentano J, Shwaery GT,
Loscalzo J and Vita JA (1993) Dietary antioxidants preserve endothelium-
dependent vessel relaxation in cholesterol-fed rabbits. Proc Natl Acad Sci USA
Send reprint requests to: Santhini Ramasamy, Ph.D., Division of Cardiol-
ogy, Emory University, 1639 Pierce Drive, Atlanta, GA 30322. E-mail:
sramasa@emory.edu
90:11880–11884.