© 1995 American Heart Association, Inc.
Articles |
Native Low-Density Lipoprotein Increases Endothelial Cell Nitric Oxide Synthase Generation of Superoxide Anion
Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993, and in full at the International Symposium on Endothelium-Derived Factors and Vascular Protection, San Francisco, Calif, January 21-25, 1995.
From the Department of Pathology (K.A.P, D.M.S.), CardioVascular Research Center, Medical College of Wisconsin, Milwaukee; the Departments of Experimental Pathology (L.G., M.W., P.V., M.B.S.) and Physiology (M.S.W.), New York Medical College, Valhalla; and Boyer Center for Molecular Medicine (W.C.S.), Yale University School of Medicine, New Haven, Conn.
Correspondence to Kirkwood A. Pritchard, Jr, PhD, Associate Professor of Pathology and Pharmacology and Toxicology, Medical College of Wisconsin, CardioVascular Research Center, Rm 494B, 8701 W Watertown Plank Rd, Milwaukee, WI 53226.
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Abstract |
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Abstract To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N
-Nitro-L-arginine methyl
ester, a specific inhibitor of eNOS activity, increases
C-EC release of O2- by >300% but decreases
LDL-treated EC (LDL-EC) release by >95%. L-Arginine
inhibits the release of O2- from LDL-ECs by
>95% but did not effect C-EC release of O2-.
Indomethacin and SKF 525A partially attenuate
LDL-induced increases in O2-
production by 
50% and 30%, respectively. Thus,
n-LDL increases O2- and NO production,
which increases the likelihood of the formation of peroxynitrite
(ONOO-), a potent oxidant. n-LDL increases the levels of
nitrotyrosine, a stable oxidation product of
ONOO-, and tyrosine by 50%. In spite of this
increase in oxidative metabolism, analysis of
thiobarbituric acid substances reveals that no significant changes in
the oxidation of n-LDL occur during the 24-hour incubations with ECs.
These data indicate that n-LDL directly perturbs
endothelial oxidative metabolism and
uncouples L-arginine metabolism from NO
release to increase eNOS generation of O2-.
Such changes may represent one of the earliest EC perturbations
in atherogenesis.
Key Words: low-density lipoprotein • reactive oxygen species • atherogenesis • peroxynitrite • superoxide anion peroxynitrite
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The relation between elevations in n-LDL concentrations and the premature development of atherosclerosis is well established. One of the primary defects in this disease process involves EC dysfunction characterized by diminished EDRF activity. Several groups have put forth the hypothesis that plasma cholesterol levels increase superoxide anion (O2-) production by the vessel wall, which inactivates EDRF.1 2 3 4 Indeed, threefold increases in this reactive species coincide with decreases in vascular relaxation during hypercholesterolemia, suggesting that inhibiting EDRF activity may play an important role in atherosclerosis.3 Although such changes may be fundamentally related to the dysfunctional states exhibited by the endothelium after exposure to n-LDL, little is known about the direct role of n-LDL in altering eNOS function.
n-LDL enhances EC prostacyclin production,5 P450-dependent generation of epoxyeicosatrienoic acids,6 endocytic activity,7 and permeability.8 In addition, n-LDL increases EC recruitment of mononuclear cells by perturbing cellular membrane lipid dynamics9 and the synthesis of adhesion molecules.10 These in vitro observations closely resemble many of the early changes observed in vivo and ex vivo in humans and animals during hypercholesterolemia.11 12 13 14
To determine the effects of n-LDL on EC reactive oxygen species generation, human umbilical vein ECs were exposed to n-LDL at 240 mg cholesterol per deciliter for 4 days, and changes in O2- and NO production were examined. n-LDL–induced changes in O2- release were assessed by the ferricytochrome c assay.15 The effects of n-LDL on NO release were examined by measuring changes in nitrite and nitrate levels by use of the Griess assay (Sessa et al16 ). Our findings indicate that n-LDL stimulates NO production and perturbs the endothelium to increase eNOS-dependent O2- production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
The reagents used for the studies were obtained as follows: medium 199 with Earle's salts, HEPES free acid, FBS, antibiotics/mycotics, heparin, potassium bromide (KBr), ferricytochrome c, SOD, sodium nitrite (NaNO2), L-arginine, L-NAME, phosphotungstate-MnCl2 precipitating reagent (catalogue No. 352-4), sucrose, Nonidet P-40, DTT, leupeptin, aprotinin, soybean trypsin inhibitor, PMSF, NADPH, CaCl2, and glucose from Sigma Chemical Co; sulfanilamide and NEDA from Aldrich; thymidine, fungizone, and DPBS) from GIBCO); Primeria 100-mm dishes and T75cm2 and T25cm2 flasks from Becton Dickinson-Falcon; six-well cluster plates from Corning; 24x30-mm Thermanox coverslips, eight-well chamber plates, and two chamber slides from Lux; human fibronectin from NY Blood Center; nonfrozen plasma from Hudson Valley Blood Services and Blood Center of Southeastern Wisconsin; EDTA from Fisher; BHT from Kodak; BH4 from Cayman Chemical Co; and, [3H]L-arginine (36.8 Ci/mol) from Dupont NEN.
Methods
EC Culture
Human umbilical vein ECs were obtained and passaged as described
previously.6 9 17 ECs were maintained in medium 199
containing 16.7% FBS, 20 mmol/L HEPES, pH 7.4, antibiotics/mycotics,
and 10 ng/mL r-bFGF). r-bFGF was kindly provided by John Anthony
Thompson (University of Alabama, Birmingham). For all studies, ECs were
used at passages 3 to 5 and grown to confluence before n-LDL exposure.
ECs were passaged onto fibronectin-coated 24x30-mm Thermanox
coverslips in eight-well chambers, six-well cluster plates, and two
chamber slides for O2-, nitrite, and
nitrotyrosine assays, respectively. Cell counts were performed on a
Coulter counter (model Zf).
Isolation of LDL and Characterization
Nonfrozen human plasma from two to five donors was obtained, and
BHT, a lipid-soluble antioxidant, and EDTA were immediately added to
the plasma to achieve final concentrations of 20 µmol/L and 0.01%,
respectively. The plasma was then mixed for 15 minutes at 4°C before
ultracentrifugation. Sterile techniques, reagents,
and dialysis solutions were used for isolation of n-LDL by sequential
density ultracentrifugation (1.019 to 1.063 g/mL)
as described previously.6 9 17 Cholesterol
levels were determined by using a cholesterol oxidase
colorimetric kit from Sigma. Endotoxin levels were
determined by the colorimetric limulus amebocyte kit
from BioWhitaker, as described previously.17 n-LDL was
stored at 4°C and used for experiments within 2 weeks. This EC model
of hypercholesterolemia was specifically
designed to examine the effects atherogenic concentrations of n-LDL on
EC function.5 6 7 9 10 17 ECs have been incubated with n-LDL
in concentrations that exceed 160 mg cholesterol per
deciliter for 4 days, with media changes every 24
hours.6 9 10 17 Previous studies demonstrated that the
protocols for protecting n-LDL with BHT essentially eliminated
nonspecific oxidation of the LDL particle during isolation and
culture.6 17 In the present study, the oxidation state
of an n-LDL particle, conditioned by standard culture with and without
ECs, was characterized by TBARS assay. TBARS analysis was
performed on conditioned C-EC and LDL-EC media as described
previously5 6 17 by first precipitating apolipoprotein B
containing lipoproteins from the media with
phosphotungstate-MnCl2. MDA equivalents were quantified on
a Cytofluor II (PerSeptive Biosystems). TBARS data were expressed as
nanomoles MDA equivalents per milligram
cholesterol.
Superoxide Anion Measurements
n-LDL–induced changes in O2- release
were determined by following increases in ferricytochrome c
absorbance at 550 nm on a Guilford spectrophotometer.15
ECs on coverslips were removed from the eight-well chamber with sterile
forceps, cut to 20x30 mm, and washed three times in three changes of
DPBS (50 mL). Two washed and trimmed coverslips were placed into a
20x30-mm quartz cuvette with the cells facing inward. DPBS (1.8 mL)
was gently pipetted into the cuvette to minimize disturbing the EC
monolayers. Ferricytochrome c (final concentration, 50
µmol/L) was added directly to the cuvette, the solution was mixed by
gentle inversion, and changes in absorbance were followed for 15
minutes. Rates of O2- production were
calculated on the basis of the molar extinction coefficient of reduced
ferricytochrome c [
=21 000
cm-1 · (mol/L)-1] and the portion that
was inhibited by SOD (400 U/mL). Cells counts were determined for
calculating results as micromoles O2- per
106 cells per minute.
To rule out the possibility that changes in O2- release develop from nonspecific reduction of ferricytochrome c, several conditions were examined. Changes in ferricytochrome c reduction were observed in the DPBS alone and also when n-LDL and ox-LDL were added directly to the ferricytochrome c/DPBS buffer. As for the EC studies above, ferricytochrome c was added directly to DPBS at a final concentration of 50 µmol/L, mixed by inversion, and placed into the spectrophotometer, and changes in absorbance were read at 550 nm for 15 minutes.
To determine the effects of L-arginine on EC O2- release, an aliquot of L-arginine (in DPBS) was pipetted directly into the assay solution (final concentration, 250 µmol/L) and mixed by inversion immediately before monitoring the changes in absorbance.
To determine which oxidative enzyme systems contribute to the release of O2- after n-LDL exposure, C-ECs and LDL-ECs were incubated in their respective media with L-NAME (final concentration, 100 µmol/L), indomethacin (final concentration, 10 µmol/L), or SKF 525A (final concentration, 30 µmol/L) for 15 minutes. The coverslips were removed and prepared for analysis as described above. All assays were carried out with the inhibitor present in the assay solutions at same concentration used for preincubation.
Total Nitrogen Oxides and Nitrite Measurements
Changes in EC production of NO were assessed by
determining nitrite levels by the Griess reaction.16 18 19
Briefly, C-ECs and LDL-ECs were washed three times with 3 mL DPBS and
then incubated with 2 mL DPBS in a 37°C oven purged with a gas
mixture of 5% CO2/95% air and rotated at 80
rpm for 30 minutes. To determine the effectiveness of L-NAME inhibition
of eNOS activity, C-ECs and LDL-ECs were incubated with L-NAME (100
µmol/L) in their respective media for 15 minutes. The cells were then
washed and incubated with DPBS containing the 100 µmol/L L-NAME as
described above for the O2- assay.
Analysis was performed on a single aliquot from each well.
Briefly, a 1 mL mixture of sulfanilamide (final reaction concentration,
0.3% [wt/vol]) and NEDA (final reaction concentration, 0.03%
[wt/vol]) was added to 0.8 mL of the conditioned DPBS, vortexed, and
then incubated for 10 minutes at 37°C. The absorbance of the
resulting pink chromophore from the unknowns was read at 550 nm and
compared with the absorbance values obtained from standard
NaNO2 solutions (0 to 200 µmol/L).
To assay for total NOx, nitrate was first converted to nitrite with an aliquot of Escherichia coli (American Type Culture Collection 25922), which was grown anaerobically to enrich nitrate reductase activity as described by Ischiropoulos et al.20 For these studies, reagents to sample volumes were adjusted so that the assay could be performed in microtiter plates. The final reagent concentration to sample volume, however, remained the same. The Griess reaction for nitrite was then carried out on aliquots of EC-conditioned media, which were preincubated with nitrate reductase–enriched E coli for 70 minutes.
eNOS Activity Measurements: The Arginine-Citrulline
Assay
The effects of n-LDL on eNOS metabolism of
[3H]arginine to [3H]citrulline were
determined as described previously.21 22 23 EC lysates were
made from C-EC and LDL-EC cultures as described
previously.21 22 23 The assay was as follows: ECs were
suspended in cold lysis buffer (0.3 mol/L sucrose, 10 mmol/L HEPES, 1%
Nonidet P-40, 0.1 mmol/L EDTA, 1 mmol/L DTT, 10 µg/mL leupeptin, 2
µg/mL aprotinin, 10 µg/mL soybean trypsin inhibitor,
and 50 µmol/L PMSF, pH 7.4), vortexed, and then kept at -80°C
until analysis. Cell lysates (150 to 250 µg protein) were
combined with NADPH (2 mmol/L), CaCl2 (230 µmol/L),
BH4 (3 µmol/L), and
[3H]L-arginine (0.2 µCi, 10 µmol/L) for
20 minutes at 37°C. The assay volume was kept constant at 100 µL.
To determine if n-LDL altered iNOS activity, the assay was repeated
with EDTA (1.7 mmol/L) replacing calcium in the assay buffers. iNOS is
calcium insensitive, whereas eNOS is calcium sensitive for
activation.
Nitrotyrosine Measurements by Enzyme-Linked
Immunoassay
ECs were grown to confluence in two chamber slides and then
incubated with n-LDL for 4 days (240 mg cholesterol per
deciliter). C-ECs and LDL-ECs were washed three times with DPBS and
then fixed in 3.7% formaldehyde in DPBS for 20 minutes. Cell membranes
were made permeable with 0.5% Triton X-100 in PBS for 15 minutes to
allow the antibodies access to intracellular proteins. Monoclonal
nitrotyrosine antibodies (kindly provided by Joseph Beckman, University
of Alabama, Birmingham) were diluted 1:50 in 1% BSA/DPBS and incubated
with the cells at room temperature for 1 hour. The slides were then
washed three times with BSA/DPBS. Next, a 1:250 dilution of anti-mouse
IgG antibody linked to horseradish peroxidase (Biorad) was added and
incubated at room temperature for 30 minutes. The slides were washed
three times with DPBS and then incubated with
OPD/H2O2 in an acetate buffer (pH 5) for 30
minutes. The reaction was stopped by adding 50 µL of 2N
H2SO4. A 200-µL aliquot of the OPD reaction
mixture was transferred to an ELISA plate, and absorbances were read at
492 nm. As a positive control for
peroxynitrite(ONOO-)–induced nitrotyrosine formation, a
second set of washed control ECs were incubated with authentic
ONOO- (2x25 µmol/L).24 Each test group, on
a separate slide, was incubated with excess nitrotyrosine (10 µmol/L)
to competitively block binding of the nitrotyrosine antibody to
cell-associated nitrotyrosine. Nonspecific antibody binding was
assessed by adding a nonspecific mouse monoclonal IgG antibody
(MOPC-31C, Sigma Chemical Co) in place of the anti-nitrotyrosine
antibody. Results are expressed as the percentage of C-EC nitrotyrosine
levels.
Statistical Analysis
Data represent the mean±SEM in the table and all
figures, unless otherwise indicated. Statistical analysis was
by the one-tailed Student's t test. The level of
significance was set at P<.05. Questions concerning the
significance of treatments were constructed to determine whether
treatments resulted in significant reductions or enhancements of
analyte production.
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Results |
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To examine the effects of atherogenic concentrations of n-LDL on EC function, it was necessary to develop isolation and culture protocols that essentially eliminated the possibility of oxidative modification of the LDL particle.5 6 17 Adding BHT to the plasma and to the sterile saline dialysis solutions is sufficient for protecting n-LDL from the oxidation observed when LDL is not protected with antioxidants.25 26 The Table
illustrates the changes in TBARS levels in n-LDL in the media incubated
in the absence and presence of ECs. TBARS analysis demonstrates
that the BHT-protected n-LDL particles experience little change in
oxidation when incubated alone or with ECs. ox-LDL is used as a control
for comparison purposes and contains 6.92 nmol MDA per milligram
cholesterol. Furthermore, n-LDL prepared under these
protocols contains levels of endotoxin that are well below those
required for activation (<0.01 EU/mL).17 Taken together,
these data demonstrate that n-LDL can be used in tissue culture for
examining the effects of hypercholesterolemia
on EC function. The protocols described here provide n-LDL in
sufficient quantities and quality to allow one to examine perturbations
in EC biochemistry before the effects of oxidized products of
altered LDL.
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Using this model of hypercholesterolemia, we
found that the n-LDL increase in EC release of
O2- is more than four times the levels
released by C-ECs (Fig 1). Results in Fig 1 are
corrected to reflect only that portion of O2-
production that is blocked by SOD (400 U/mL). The majority of
O2- signal from both C-ECs and LDL-ECs is
blocked by SOD (C-EC+SOD, 0.026±0.004 µmol
O2- per minute per 106
cells; LDL-EC+SOD, 0.031±0.006 µmol O2- per
minute per 106 cells). Furthermore, BHT did not
appear to interfere with the ability of endothelium to
release O2-. Thus, atherogenic concentrations
of n-LDL induce profound changes in the oxidative
metabolism of the endothelium. No increases
in ferricytochrome c absorbances were noted in DPBS or when
n-LDL (20 µg protein per milliliter) or ox-LDL (20 µg protein per
milliliter) was added to the assay buffers (data not shown). Our
findings demonstrate that the increases in O2-
release are the direct result of n-LDL effects on the
endothelium and are not due to an artifact of ox-LDL in
the assay system.
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Several mechanisms are proposed to explain increases in
O2- production of the vessel wall
during hypercholesterolemia.3 4 27
Loss of functional NO is examined by including L-arginine
or L-NAME in the buffers of the ferricytochrome c assay.
L-Arginine (250 µmol/L) does not significantly alter
C-EC release of O2- (Fig 1
). In contrast,
L-arginine markedly decreases the release of
O2- from LDL-ECs to the levels obtained with
SOD or L-NAME. Since L-arginine does not inhibit
xanthine/xanthine oxidase reduction of ferricytochrome
c,28 it is likely that reductions in LDL-EC
O2- release develop from uptake and
metabolism of this amino acid to NO, which in turn
scavenges O2-. Alternatively, excess
L-arginine may keep the heme site of eNOS fully occupied
to limit uncoupled O2- generation. Regardless
of the mechanism, adding L-arginine reduces
O2- production rates by LDL-ECs below
the levels observed for C-ECs.
To determine whether NO plays equivalent roles in C-EC and LDL-EC
cultures, the effects of L-NAME, a specific inhibitor of
NOS activity, on O2- release are assessed.
L-NAME increases C-EC release of O2- by
>300% (Fig 1). These data are consistent with the notion that
NO functions as an intracellular scavenger of
O2- and that decreases in functional NO
increase the release of O2-. In contrast,
L-NAME reduces LDL-EC release of O2- by >95%
(Fig 1). According to our previous studies, n-LDL increases COX and
P450 activities,6 29 which are well-recognized sources of
O2- production.30 To
examine the contribution of these pathways, C-ECs and LDL-ECs are
incubated with indomethacin (10 µmol/L) and SKF 525A
(30 µmol/L). Indomethacin and SKF 525A reduce LDL-EC
release rates by 50% and 30%, respectively (Fig 1). However, it is
important to note that L-NAME is more effective in inhibiting LDL-EC
O2- release than are
indomethacin and SKF 525A (Fig 1). Thus, protracted EC
exposure to atherogenic n-LDL concentrations increases
O2- production by three independent
oxidative enzyme systems. More important, eNOS appears to be the
greatest source among these pathways for n-LDL-induced increases in
O2- production.
To determine whether n-LDL alters the usual generation of NO, the
levels of NOx released into 24-hour conditioned culture
media from day 4 and the levels of nitrite released into 30-minute
conditioned DPBS were determined.16 18 19 During culture
in medium 199, which contains 330 µmol/L L-arginine,
n-LDL increases NOx levels by 20-fold (Fig 2A). In addition, in the absence of
L-arginine, NO2-
production rates by washed LDL-ECs are significantly increased
(Fig 2B), although the relative increase is much smaller than when ECs
are exposed to media containing both n-LDL and L-arginine
(Fig 2A). BHT did not appear to inhibit the ability of
endothelium to generate NOx or
NO2-. In our hands, adding L-NAME to cultures
inhibits formation of 50% of the chromophore detected by the Griess
reaction (data not shown).
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The effects of n-LDL on eNOS activity are examined by the
[3H]citrulline assay, which is performed under apparent
Vmax conditions.21 22 23 n-LDL did not
significantly alter the ability of eNOS in lysed cell preparations to
metabolize L-arginine to L-citrulline
(81.4±11.7 [C-ECs] versus 56.8±15.3 [LDL-ECs] pmol
citrulline per milligram protein per minute, n=4, mean±SD).
Furthermore, the activity of iNOS, a calcium-independent enzyme, in
these cell preparations is sufficiently low (0.45 pmol citrulline
per milligram protein per minute) that it is unlikely that it could
account for n-LDL–induced increases in reactive oxygen species
generation.
Dual increases in O2- and NO are the
prerequisite conditions for increasing ONOO-
formation.31 32 ONOO- oxidizes tyrosine to
nitrotyrosine, which can be detected immunogenically.4 32
To determine the effects of n-LDL on ONOO- formation,
nitrotyrosine levels were measured by using antibodies kindly provided
by Joseph Beckman (Birmingham, Ala). n-LDL significantly increases
immunoreactive nitrotyrosine levels by 50% above C-EC levels. The
nitrotyrosine levels in LDL-EC cultures after 4 days are 33% of the
levels formed after exposing C-ECs to two doses of authentic
ONOO- (Fig 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates that pathophysiological concentrations of n-LDL perturb EC oxidative metabolism to increase O2- production. NO plays an important role in limiting the amount of O2- released by ECs, as illustrated by L-NAME–induced increases in O2- release from C-ECs. However, inhibition of O2- release from LDL-ECs by L-NAME points to a mechanism by which n-LDL perturbs EC function to increase eNOS generation of O2-. Selective inhibition of COX and P450 isozymes attenuates increases in O2- release induced by n-LDL but not to the extent that L-NAME inhibits O2-. Thus, n-LDL increases oxidative metabolism by several enzyme systems. However, of the enzyme systems examined, eNOS appears to generate the largest portion of the O2-. This conclusion is supported by decreases in O2- release from LDL-ECs but not C-ECs with 250 µmol/L L-arginine. These data suggest that in LDL-EC cultures L-arginine metabolism may be impaired. Further, uncoupling L-arginine metabolism from NO release is probably a critical step in eNOS-dependent O2- generation. The ability of the endothelium to produce NO is not compromised, since n-LDL, in the presence of L-arginine, stimulates increases in NO production. Furthermore, the conversion of [3H]arginine into [3H]citrulline under apparent Vmax conditions, a measure of eNOS levels, is not significantly different between LDL-EC and C-EC lysates. BHT did not appear to inhibit O2- and NOx synthesis, since rates of production of these two analytes were not impaired. Dual increases in the generation of O2- and NO are the principal and prerequisite conditions for ONOO- formation, which is increased in LDL-ECs. Thus, protracted n-LDL exposure induces EC dysfunction such that eNOS becomes a new source for O2- generation and ONOO- formation. These studies link atherogenic concentrations of n-LDL with increases in O2- generation from altered eNOS activity.
The central hypothesis of our laboratories is that n-LDL directly
alters endothelial function.5 6 7 9 17
Previous studies indicate that n-LDL prepared by our methods does not
experience significant increases in oxidation.6 17 Thus,
adding BHT to the plasma before n-LDL isolation is sufficient to limit
oxidation during culture. Protecting n-LDL against oxidation is the
first step in being able to examine perturbations in EC function
induced by n-LDL rather than by ox-LDL. Our new findings extend
previous studies by demonstrating how n-LDL uncouples
L-arginine metabolism from eNOS activity,
which in turn alters one of the critical functions of the
endothelium generation of EC-derived NO. n-LDL
uncouples L-arginine metabolism from eNOS
generation of NO, resulting in increased O2-
and ONOO- production. Under these conditions,
functional NO levels are probably diminished as well. Thus,
n-LDL–exposed ECs will experience not only increases in
O2- production but also decreases in
NO. Such perturbations in usual reactive oxygen species generation may
play an important role in the activation of oxidant-sensitive
transcription factors, ie, nuclear factor-
B and fos/jun,
which in turn influence the expression of adhesion
molecules.33 34 35 Interestingly, when L-NAME is used to
inhibit NO production in human endothelial
cells, intercellular adhesion molecule-1–dependent binding of
neutrophils is increased.36 The effects of n-LDL on
O2- and ONOO- generation may
explain, in part, the mechanisms by which n-LDL increases
endothelial adherence of mononuclear cells observed by
us in vitro9 17 and by Tsao et al28 ex vivo.
Interestingly, L-arginine suppression of
O2- release from LDL-EC cultures reveals new
insight into a possible mechanism by which L-arginine
supplementation of cholesterol-fed rabbits decreases
mononuclear adherence.28
The relative levels of NO generation by cells of the vessel wall are critical for proper function of the vasculature. During hypercholesterolemia, the vessel wall experiences changes in the balance of oxidative and antioxidative enzyme systems, resulting in increases in O2- release.3 37 Early in the disease state, this increase is manifested in the endothelium3 ; later, it is predominantly associated with the monocytes/macrophages, which release reactive oxygen species in much larger concentrations than ECs.20 38 Inhibition of eNOS with arginine analogues not only compromises endothelium-dependent mechanisms of relaxation but also perturbs the balance between oxidation and reduction.28 39 40 41 Such changes appear intimately linked to increases in plaque development.28 39 40 41 From the above studies, one can conclude that NO functions as an antiatherogenic molecule, as suggested by Cooke and Tsao.42
Recent evidence indicates that when cellular levels of O2- and NO are increased, the potent oxidant, ONOO-, is quickly formed.20 31 32 ONOO- is described as a "cloaked oxidant," which exhibits a wide range of oxidative cytotoxic properties.31 Its pathophysiological importance was recently demonstrated by using nitrotyrosine antibodies. In these studies, advanced plaques were found to contain high levels of nitrotyrosine, implicating involvement of ONOO- in late- to end-stage lesion formation.4 43
ONOO- formation and its relevance to atherosclerosis should not, however, be limited to mononuclear type cells. When ECs are stimulated with ionophore, marked increases in ONOO- are observed.44 45 Our data indicate that protracted EC exposure to atherogenic LDL concentrations significantly increases EC production of ONOO- during basal metabolic activity. Thus, ONOO--mediated atherogenic mechanisms are probably invoked before the oxidant injury mechanisms in advanced lesions. These findings link n-LDL with increases in endothelial ONOO- generation. Accordingly, such changes will play critical roles in endothelial activation mechanisms that are oxidant stress sensitive.
Increases in NO production induced by n-LDL indicate that this lipoprotein acts as a stimulant for eNOS activity. Increases in ONOO- induced by n-LDL result from NO condensing with O2-, an event that not only lowers functional NO levels but also increases the formation of a new oxidant with properties different from either O2- or NO. These in vitro findings support the concept that although hypercholesterolemia increases NO production early in the atherogenic process, the NO that is produced is ineffective.27 Because [3H]citrulline assays are performed under apparent Vmax conditions, the conversion rates are representative of enzyme levels. Although rates of conversion in LDL-EC lysates are slightly less than the rates in C-EC lysates, these differences are not significant. Together, these data suggest that n-LDL may perturb either substrate delivery or the availability of a cofactor to promote eNOS generation of O2-. It should be noted that the ferricytochrome c assay did not include L-arginine, a condition that is not optimal for eNOS activity. Perhaps the absence of L-arginine magnifies defects in eNOS function, leading to increases in O2- release from LDL-ECs. In vivo evidence that eNOS can be uncoupled to increase oxidant stress is found in rabbits fed diaminohydroxypyrimidine to block synthesis of BH4.46 This treatment results in aortas that produce higher levels of H2O2, a product of O2- dismutation, than do control aortas. Finally, the effects of n-LDL on eNOS are opposite those of LDL that oxidizes during culture26 and ox-LDL47 but are consistent with increases in NOx production by aortas from cholesterol-fed rabbits.27 Thus, n-LDL uncoupling of eNOS could be viewed as a mechanism that occurs early in the atherogenic process, whereas the effects of ox-LDL could be viewed as a series of mechanisms that occur after LDL enters the vessel wall and becomes trapped and then modified.
L-NAME and L-arginine inhibition of eNOS O2- generation are both significant and complementary. One agent blocks the activity of eNOS; the other supplies excess substrate for generating NO, which in turn scavenges O2-.27 48 Indomethacin and SKF 525A do not lower LDL-EC O2- production to control levels, because eNOS continues to generate O2-. The levels of indomethacin and SKF 525A used here completely block arachidonic acid metabolism by COX and P450 isozymes, respectively,6 but do not alter eNOS activity. L-NAME does not inhibit the metabolism of arachidonic acid by COX and P450 isozymes (authors' unpublished observations, 1994). On the basis of the specificity of L-NAME and L-arginine for eNOS and the fact that reciprocal changes in O2- release are observed after treatment, it is unlikely the increase in O2- is derived from non-eNOS sources that are sensitive to both agents in the same way. We acknowledge that our data do not exclude such a possibility but consider it to be extremely remote.
At first glance, data in Fig 1 may appear inconsistent,
since inhibitions by L-NAME, indomethacin, and SKF 525A
are greater than 100%. The reason this occurs is not clear.
Extracellular release of O2- from ECs
represents the sum totals of generation and scavenging as well
as the effects of compartmentation and diffusion. Further, changes in
NO and O2- production by eNOS after
n-LDL treatments may confer unknown effects on COX and P450 activities.
For example, NO increases COX prostaglandin
production49 and inhibits P450
activity.50 Thus, NO from L-arginine may
play a role in increasing O2- by enhancing COX
activity. On the other hand, loss of functional NO, which likely occurs
in LDL-ECs, may allow P450-dependent O2-
production to increase, because NO is not available to inhibit
the heme site of P450 isozymes. However, the effects of NO on
O2- generation from these two sources are not
well studied. Finally, LDL-induced increases in
O2- and ONOO- may enhance the
conversion of xanthine dehydrogenase to xanthine oxidase to increase
O2-.51 This possibility may
explain the mechanism by which xanthine oxidase in the aortas of
cholesterol-fed rabbits became a source of
O2- production.3
Unfortunately, because of the complexities of the interaction of NO
with COX and P450 (and possibly other oxidative enzymes) it may not be
possible to sort out the exact contribution each oxidative pathway
makes by using inhibitors.
Another problem facing studies designed to probe the role of oxidative enzymes in reactive oxygen generation is inhibitor specificity. Indomethacin and SKF 525A are considered selective inhibitors of COX and P450 isozymes, yet these agents may alter EC function in ways that go beyond their direct effects on enzyme activity. For instance, it has been reported that indomethacin is capable of scavenging O2- and that SKF 525A is capable of perturbing calcium homeostasis.52 53 For our studies, we chose concentrations of indomethacin and SKF 525A that were previously shown not to scavenge O2-38 ; accordingly, the decreases in O2- detected should represent the relative contribution these pathways make to the total signal induced by n-LDL. Inhibitor targeting of other oxidative enzymes also has its problems. For example, diphenylene iodonium, a selective inhibitor of NADPH oxidoreductase, also inhibits NOS.54 Accordingly, the effects of this agent on both enzymes would further confuse the issue of which and how much each source contributes to n-LDL–induced increases in O2- generation. However, it should be noted that L-NAME is probably the most specific inhibitor of the agents used in the present study. Further, when L-NAME and L-arginine data are examined together, it strongly suggests that n-LDL uncouples eNOS activity to induce this enzyme to generate O2-.
The mechanisms by which L-NAME and L-arginine analogues specifically modulate brain NOS O2- generation are in dispute at this time. Pou et al55 show that L-NAME but not NG-monomethyl-L-arginine blocks neuronal brain NOS O2- production as did Heinzel et al56 for neuronal NOS H2O2 production. Frey et al57 report that L-thiocitrulline, a new inhibitor of NOS activity, blocks nearly 85% of the O2- signal from neuronal brain NOS under uncoupling conditions. Possibly, some insight into the mechanisms by which L-NAME inhibits O2- production can be obtained from the knowledge that L-NAME binds within the active site of NOS and decreases electron flux by inhibiting the reduction potential of heme iron.58 These studies suggest that L-NAME inhibits NOS O2- generation because it prevents electron transfer to the heme site. L-NMMA inhibits NOS conversion of arginine to citrulline by occupying the active site but does not appear to block electron transfer to the heme site. If this is the case, then it is likely that the iron heme group becomes electron rich and transfers the electrons to molecular oxygen to generate O2-. An alternative mechanism for O2- generation from NOS, however, may involve BH4; this finding is based on work demonstrating that neuronal brain NOS, made deficient in BH4, concurrently generates O2-, H2O2, and NO when L-arginine is present in low levels, a finding that can be fully reversed by adding BH4.56 Regardless of the mechanisms by which NOS generates O2-, data presented here clearly indicate that n-LDL uncouples eNOS activity to increase O2- production in L-arginine–deficient buffers.
The mechanisms by which n-LDL perturbs eNOS function to increase O2- generation remain unclear. In vitro and in vivo studies suggest that uncoupled O2- generation may be a unique property of eNOS that is manifested under conditions of L-arginine depletion and/or lack of cofactors such as BH4. When electron transfer is uncoupled in oxygenase enzymes, oxygen becomes the usual electron acceptor, resulting in O2- generation.59 When L-arginine is left out of brain NOS assays, increases in O2- production are observed.55 In vitro, the reduction of ferricytochrome c by neuronal brain NOS, in the presence of calcium/calmodulin, can be substantially reduced by SOD.57 60 When L-arginine levels fall below 100 µmol/L, NADPH oxidation is uncoupled from NOS synthesis of NO,56 which will increase O2- production. Under other conditions, glutamate uncouples L-arginine metabolism from neuronal NOS NO generation and increases in O2- production.61 BH4-deficient NOS concurrently generates O2-, H2O2, and NO; however, when BH4 is provided, maximal NO production is restored.56 This last mechanism may explain the increase in H2O2 production in aortas from rabbits fed diaminohydroxypyrimidine,46 an inhibitor of GTP cyclohydrolase I, the first enzyme in the synthesis of BH4.62 Taken together, these reports suggest that uncoupling L-arginine metabolism from NOS activity leads to increased O2- in vitro and in vivo. Previously, we demonstrated that n-LDL increases EC membrane rigidity through donating cholesterol.9 Changes in membrane lipid dynamics might impede the binding, internalization, and delivery of L-arginine to eNOS. Hypothetically, if n-LDL enhances eNOS activity and impedes the process governing L-arginine delivery to NOS, then intracellular L-arginine concentrations surrounding NOS may become depleted. According to the information above, such a state might uncouple NOS activity and increase the generation of O2-. Thus, one potential mechanism by which n-LDL may be inducing eNOS to generate O2- is by decreasing membrane fluidity to perturb usual L-arginine metabolism. Other potential mechanisms for the effects of n-LDL include perturbations in BH4 metabolism and calcium/calmodulin interactions. Finally, n-LDL may perturb the basic enzymatic properties of eNOS such that this trifunctional enzyme exhibits different requirements for L-arginine, BH4, flavin adenine dinucleotide, flavin mononucleotide, and /or calcium/calmodulin.
In summary, our findings demonstrate that n-LDL alters the usual reactive oxygen species generation. More important, L-NAME and L-arginine inhibition of O2- release from LDL-EC supports the notion that n-LDL uncouples L-arginine metabolism from NO release, thereby allowing eNOS to become a new source of O2-. These findings provide new insight into the mechanisms by which n-LDL both stimulates NO production and decreases functional NO levels through the formation of ONOO- (L-arginine supplementation limits n-LDL–induced mechanisms of endothelial dysfunction) and demonstrate the importance of NO limiting O2- release. These studies demonstrate that protracted EC exposure to atherogenic concentrations of n-LDL uncouples L-arginine metabolism from NO production and increases eNOS-dependent O2- generation and ONOO- formation. Finally, such perturbations may be central to n-LDL–induced mechanisms of EC dysfunction and atherogenesis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Heart, Lung, and Blood Institute grants HL-48521, HL-33742, HL-31069, and 1-PO HL-43023R1 and grant 93-006GB from the American Heart Association, New York State Affiliate, Inc. We gratefully acknowledge the gift of anti-nitrotyrosine antibodies kindly provided by Dr Joseph Beckman (University of Alabama, Birmingham) and extend our thanks to Drs Kalyanaraman, Hogg, and Griffith (Medical College of Wisconsin, Milwaukee) for their advice and comments in the writing of this manuscript. We also thank the staff of Labor and Delivery at White Plains Hospital (White Plains, NY) and Waukeshaw Memorial Hospital (Waukeshaw, Wis) for providing umbilical cords and Hudson Valley Blood Center (Valhalla, NY) and the Blood Center of Southeastern Wisconsin (Milwaukee) for providing plasma used for these studies. Finally, we thank Michelle Italiano for technical assistance in culturing of endothelial cells and n-LDL isolation.
Received September 26, 1994; accepted May 8, 1995.
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J. Ou, J. T. Fontana, Z. Ou, D. W. Jones, A. W. Ackerman, K. T. Oldham, J. Yu, W. C. Sessa, and K. A. Pritchard Jr. Heat shock protein 90 and tyrosine kinase regulate eNOS NO{middle dot} generation but not NO{middle dot} bioactivity Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H561 - H569. [Abstract] [Full Text] [PDF]
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T. Thum and J. Borlak Mechanistic Role of Cytochrome P450 Monooxygenases in Oxidized Low-Density Lipoprotein-Induced Vascular Injury: Therapy Through LOX-1 Receptor Antagonism? Circ. Res., January 9, 2004; 94 (1): e1 - e13. [Abstract] [Full Text] [PDF]
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M. I. Lin, D. Fulton, R. Babbitt, I. Fleming, R. Busse, K. A. Pritchard Jr., and W. C. Sessa Phosphorylation of Threonine 497 in Endothelial Nitric-oxide Synthase Coordinates the Coupling of L-Arginine Metabolism to Efficient Nitric Oxide Production J. Biol. Chem., November 7, 2003; 278(45): 44719 - 44726. [Abstract] [Full Text] [PDF]
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J. P. Cooke, K. Sydow, J. Chen, and P. Huang A Peculiar Result and a Fanciful Hypothesis Regarding L-Arginine * Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 1128 - 1128. [Full Text] [PDF]
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M. Weis and J. P. Cooke Cardiac Allograft Vasculopathy and Dysregulation of the NO Synthase Pathway Arterioscler Thromb Vasc Biol, April 1, 2003; 23(4): 567 - 575. [Abstract] [Full Text] [PDF]
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Z. Ou, J. Ou, A. W. Ackerman, K. T. Oldham, and K. A. Pritchard Jr L-4F, an Apolipoprotein A-1 Mimetic, Restores Nitric Oxide and Superoxide Anion Balance in Low-Density Lipoprotein-Treated Endothelial Cells Circulation, March 25, 2003; 107(11): 1520 - 1524. [Abstract] [Full Text] [PDF]
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F. de Nigris, L. O. Lerman, S. W. Ignarro, G. Sica, A. Lerman, W. Palinski, L. J. Ignarro, and C. Napoli From the Cover: Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress PNAS, February 4, 2003; 100(3): 1420 - 1425. [Abstract] [Full Text] [PDF]
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P. Piatti, G. Fragasso, L. D. Monti, E. Setola, P. Lucotti, I. Fermo, R. Paroni, E. Galluccio, G. Pozza, S. Chierchia, et al. Acute Intravenous l-Arginine Infusion Decreases Endothelin-1 Levels and Improves Endothelial Function in Patients With Angina Pectoris and Normal Coronary Arteriograms: Correlation With Asymmetric Dimethylarginine Levels Circulation, January 28, 2003; 107(3): 429 - 436. [Abstract] [Full Text] [PDF]
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T. Iuchi, M. Akaike, T. Mitsui, Y. Ohshima, Y. Shintani, H. Azuma, and T. Matsumoto Glucocorticoid Excess Induces Superoxide Production in Vascular Endothelial Cells and Elicits Vascular Endothelial Dysfunction Circ. Res., January 10, 2003; 92(1): 81 - 87. [Abstract] [Full Text] [PDF]
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G. E. Mann, D. L. Yudilevich, and L. Sobrevia Regulation of Amino Acid and Glucose Transporters in Endothelial and Smooth Muscle Cells Physiol Rev, January 1, 2003; 83(1): 183 - 252. [Abstract] [Full Text] [PDF]
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A. C. Sposito and M. J. Chapman Statin Therapy in Acute Coronary Syndromes: Mechanistic Insight Into Clinical Benefit Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1524 - 1534. [Abstract] [Full Text] [PDF]
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A. Sato, H. Miura, Y. Liu, L. B. Somberg, M. F. Otterson, M. J. Demeure, W. J. Schulte, L. M. Eberhardt, F. R. Loberiza, I. Sakuma, et al. Effect of gender on endothelium-dependent dilation to bradykinin in human adipose microvessels Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H845 - H852. [Abstract] [Full Text] [PDF]
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D. W. Stepp, J. Ou, A. W. Ackerman, S. Welak, D. Klick, and K. A. Pritchard Jr. Native LDL and minimally oxidized LDL differentially regulate superoxide anion in vascular endothelium in situ Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H750 - H759. [Abstract] [Full Text] [PDF]
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R. W. van Etten, E. J.P. de Koning, M. L. Honing, E. S. Stroes, C. A. Gaillard, and T. J. Rabelink Intensive Lipid Lowering by Statin Therapy Does Not Improve Vasoreactivity in Patients With Type 2 Diabetes Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 799 - 804. [Abstract] [Full Text] [PDF]
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M. A.W. Broeders, G. J. Tangelder, D. W. Slaaf, R. S. Reneman, and M. G.A. oude Egbrink Hypercholesterolemia Enhances Thromboembolism in Arterioles but Not Venules: Complete Reversal by L-Arginine Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 680 - 685. [Abstract] [Full Text] [PDF]
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H. Mollnau, M. Wendt, K. Szocs, B. Lassegue, E. Schulz, M. Oelze, H. Li, M. Bodenschatz, M. August, A. L. Kleschyov, et al. Effects of Angiotensin II Infusion on the Expression and Function of NAD(P)H Oxidase and Components of Nitric Oxide/cGMP Signaling Circ. Res., March 8, 2002; 90 (4): e58 - e65. [Abstract] [Full Text] [PDF]
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D. M. Lenda and M. A. Boegehold Effect of a high-salt diet on oxidant enzyme activity in skeletal muscle microcirculation Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H395 - H402. [Abstract] [Full Text] [PDF]
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M.C. Verhaar, E. Stroes, and T.J. Rabelink Folates and Cardiovascular Disease Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 6 - 13. [Abstract] [Full Text] [PDF]
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Y.-M. Go, A.-L. Levonen, D. Moellering, A. Ramachandran, R. P. Patel, H. Jo, and V. M. Darley-Usmar Endothelial NOS-dependent activation of c-Jun NH2- terminal kinase by oxidized low-density lipoprotein Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2705 - H2713. [Abstract] [Full Text] [PDF]
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G. Wiemer, G. Itter, T. Malinski, and W. Linz Decreased Nitric Oxide Availability in Normotensive and Hypertensive Rats With Failing Hearts After Myocardial Infarction Hypertension, December 1, 2001; 38(6): 1367 - 1371. [Abstract] [Full Text] [PDF]
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A. J. Cayatte, A. Rupin, J. Oliver-Krasinski, K. Maitland, P. Sansilvestri-Morel, M.-F. Boussard, M. Wierzbicki, T. J. Verbeuren, and R. A. Cohen S17834, a New Inhibitor of Cell Adhesion and Atherosclerosis That Targets NADPH Oxidase Arterioscler Thromb Vasc Biol, October 1, 2001; 21(10): 1577 - 1584. [Abstract] [Full Text] [PDF]
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N. Wang, L. Verna, H.-l. Liao, A. Ballard, Y. Zhu, and M. B. Stemerman Adenovirus-Mediated Overexpression of Dominant-Negative Mutant of c-Jun Prevents Intercellular Adhesion Molecule-1 Induction by LDL: A Critical Role for Activator Protein-1 in Endothelial Activation Arterioscler Thromb Vasc Biol, September 1, 2001; 21(9): 1414 - 1420. [Abstract] [Full Text] [PDF]
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J. S. Beckman -OONO: Rebounding From Nitric Oxide Circ. Res., August 17, 2001; 89(4): 295 - 297. [Full Text] [PDF]
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H. A. Walker, E. McGing, I. Fisher, R. H. Boger, S. M. Bode-Boger, G. Jackson, J. M. Ritter, and P. J. Chowienczyk Endothelium-dependent vasodilation is independent of the plasma L-arginine/ADMA ratio in men with stable angina: Lack of effect of oral l-arginine on endothelial function, oxidative stress and exercise performance J. Am. Coll. Cardiol., August 1, 2001; 38(2): 499 - 505. [Abstract] [Full Text] [PDF]
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F. Cosentino, J. E. Barker, M. P. Brand, S. J. Heales, E. R. Werner, J. R. Tippins, N. West, K. M. Channon, M. Volpe, and T. F. Luscher Reactive Oxygen Species Mediate Endothelium-Dependent Relaxations in Tetrahydrobiopterin-Deficient Mice Arterioscler Thromb Vasc Biol, April 1, 2001; 21(4): 496 - 502. [Abstract] [Full Text] [PDF]
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J. A. Beckman, A. B. Goldfine, M. B. Gordon, and M. A. Creager Ascorbate Restores Endothelium-Dependent Vasodilation Impaired by Acute Hyperglycemia in Humans Circulation, March 27, 2001; 103(12): 1618 - 1623. [Abstract] [Full Text] [PDF]
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J. B. Laursen, M. Somers, S. Kurz, L. McCann, A. Warnholtz, B. A. Freeman, M. Tarpey, T. Fukai, and D. G. Harrison Endothelial Regulation of Vasomotion in ApoE-Deficient Mice : Implications for Interactions Between Peroxynitrite and Tetrahydrobiopterin Circulation, March 6, 2001; 103(9): 1282 - 1288. [Abstract] [Full Text] [PDF]
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R. H. Boger, S. M. Bode-Boger, P. S. Tsao, P. S. Lin, J. R. Chan, and J. P. Cooke An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2287 - 2295. [Abstract] [Full Text] [PDF]
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M. Oelze, H. Mollnau, N. Hoffmann, A. Warnholtz, M. Bodenschatz, A. Smolenski, U. Walter, M. Skatchkov, T. Meinertz, and T. Munzel Vasodilator-Stimulated Phosphoprotein Serine 239 Phosphorylation as a Sensitive Monitor of Defective Nitric Oxide/cGMP Signaling and Endothelial Dysfunction Circ. Res., November 24, 2000; 87(11): 999 - 1005. [Abstract] [Full Text] [PDF]
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J. P. Cooke Does ADMA Cause Endothelial Dysfunction? Arterioscler Thromb Vasc Biol, September 1, 2000; 20(9): 2032 - 2037. [Abstract] [Full Text] [PDF]
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K. K. Koh Effects of statins on vascular wall: vasomotor function, inflammation, and plaque stability Cardiovasc Res, September 1, 2000; 47(4): 648 - 657. [Abstract] [Full Text] [PDF]
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D. Tomasian, J. F. Keaney Jr., and J. A. Vita Antioxidants and the bioactivity of endothelium-derived nitric oxide Cardiovasc Res, August 18, 2000; 47(3): 426 - 435. [Full Text] [PDF]
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K. M. Channon, H. Qian, and S. E. George Nitric Oxide Synthase in Atherosclerosis and Vascular Injury : Insights From Experimental Gene Therapy Arterioscler Thromb Vasc Biol, August 1, 2000; 20(8): 1873 - 1881. [Abstract] [Full Text] [PDF]
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R. H. Boger, K. Sydow, J. Borlak, T. Thum, H. Lenzen, B. Schubert, D. Tsikas, and S. M. Bode-Boger LDL Cholesterol Upregulates Synthesis of Asymmetrical Dimethylarginine in Human Endothelial Cells : Involvement of S-Adenosylmethionine-Dependent Methyltransferases Circ. Res., July 21, 2000; 87(2): 99 - 105. [Abstract] [Full Text] [PDF]
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P. A. Kaufmann, T. Gnecchi-Ruscone, K. P. Schafers, T. F. Luscher, and P. G. Camici Low density lipoprotein cholesterol and coronary microvascular dysfunction in hypercholesterolemia J. Am. Coll. Cardiol., July 1, 2000; 36(1): 103 - 109. [Abstract] [Full Text] [PDF]
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E. S. G. Stroes, E. E. van Faassen, M. Yo, P. Martasek, P. Boer, R. Govers, and T. J. Rabelink Folic Acid Reverts Dysfunction of Endothelial Nitric Oxide Synthase Circ. Res., June 9, 2000; 86(11): 1129 - 1134. [Abstract] [Full Text] [PDF]
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M. S. Wolin Interactions of Oxidants With Vascular Signaling Systems Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1430 - 1442. [Abstract] [Full Text] [PDF]
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U. Landmesser, R. Merten, S. Spiekermann, K. Buttner, H. Drexler, and B. Hornig Vascular Extracellular Superoxide Dismutase Activity in Patients With Coronary Artery Disease : Relation to Endothelium-Dependent Vasodilation Circulation, May 16, 2000; 101(19): 2264 - 2270. [Abstract] [Full Text] [PDF]
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T. J. Guzik, N. E. J. West, E. Black, D. McDonald, C. Ratnatunga, R. Pillai, and K. M. Channon Vascular Superoxide Production by NAD(P)H Oxidase : Association With Endothelial Dysfunction and Clinical Risk Factors Circ. Res., May 12, 2000; 86 (9): e85 - e90. [Abstract] [Full Text] [PDF]
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K. Kauser, V. da Cunha, R. Fitch, C. Mallari, and G. M. Rubanyi Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1679 - H1685. [Abstract] [Full Text] [PDF]
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L. Vergnani, S. Hatrik, F. Ricci, A. Passaro, N. Manzoli, G. Zuliani, V. Brovkovych, R. Fellin, and T. Malinski Effect of Native and Oxidized Low-Density Lipoprotein on Endothelial Nitric Oxide and Superoxide Production : Key Role of L-Arginine Availability Circulation, March 21, 2000; 101(11): 1261 - 1266. [Abstract] [Full Text] [PDF]
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L. P. Perrault, F. Mahlberg, C. Breugnot, J.-P. Bidouard, N. Villeneuve, J.-P. Vilaine, and P. M. Vanhoutte Hypercholesterolemia Increases Coronary Endothelial Dysfunction, Lipid Content, and Accelerated Atherosclerosis After Heart Transplantation Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 728 - 736. [Abstract] [Full Text] [PDF]
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R. Golser, A. C. F. Gorren, A. Leber, P. Andrew, H.-J. Habisch, E. R. Werner, K. Schmidt, R. C. Venema, and B. Mayer Interaction of Endothelial and Neuronal Nitric-oxide Synthases with the Bradykinin B2 Receptor. BINDING OF AN INHIBITORY PEPTIDE TO THE OXYGENASE DOMAIN BLOCKS UNCOUPLED NADPH OXIDATION J. Biol. Chem., February 25, 2000; 275(8): 5291 - 5296. [Abstract] [Full Text] [PDF]
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T. Munzel, H. Li, H. Mollnau, U. Hink, E. Matheis, M. Hartmann, M. Oelze, M. Skatchkov, A. Warnholtz, L. Duncker, et al. Effects of Long-Term Nitroglycerin Treatment on Endothelial Nitric Oxide Synthase (NOS III) Gene Expression, NOS III-Mediated Superoxide Production, and Vascular NO Bioavailability Circ. Res., January 7, 2000; 86 (1): e7 - e12. [Abstract] [Full Text] [PDF]
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A. A. Ogonowski, W. H. Kaesemeyer, L. Jin, V. Ganapathy, F. H. Leibach, and R. W. Caldwell Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production Am J Physiol Cell Physiol, January 1, 2000; 278(1): C136 - C143. [Abstract] [Full Text] [PDF]
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C. Palombo, V. Lubrano, and T. Sampietro Oxidative stress, F2-isoprostanes and endothelial dysfunction in hypercholesterolemia Cardiovasc Res, December 1, 1999; 44(3): 474 - 476. [Full Text] [PDF]
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D. D. Gutterman Adventitia-dependent influences on vascular function Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1265 - H1272. [Full Text] [PDF]
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M. McIntyre, D. F. Bohr, and A. F. Dominiczak Endothelial Function in Hypertension : The Role of Superoxide Anion Hypertension, October 1, 1999; 34(4): 539 - 545. [Abstract] [Full Text] [PDF]
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P. J Andrew and B. Mayer Enzymatic function of nitric oxide synthases Cardiovasc Res, August 15, 1999; 43(3): 521 - 531. [Abstract] [Full Text] [PDF]
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I. Fleming and R. Busse Signal transduction of eNOS activation Cardiovasc Res, August 15, 1999; 43(3): 532 - 541. [Abstract] [Full Text] [PDF]
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W. Linz, P. Wohlfart, B. A Scholkens, T. Malinski, and G. Wiemer Interactions among ACE, kinins and NO Cardiovasc Res, August 15, 1999; 43(3): 549 - 561. [Full Text] [PDF]
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H. Drexler Nitric oxide and coronary endothelial dysfunction in humans Cardiovasc Res, August 15, 1999; 43(3): 572 - 579. [Full Text] [PDF]
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G. Kojda and D. Harrison Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure Cardiovasc Res, August 15, 1999; 43(3): 652 - 671. [Full Text] [PDF]
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F. Cosentino and T. F Luscher Tetrahydrobiopterin and endothelial nitric oxide synthase activity Cardiovasc Res, August 1, 1999; 43(2): 274 - 278. [Full Text] [PDF]
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J. R. Kersten and D. C. Warltier Modulation of the adaptive response to myocardial ischemia by coexisting disease Am J Physiol Heart Circ Physiol, June 1, 1999; 276(6): H2268 - H2270. [Full Text] [PDF]
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K. Kusterer, T. Pohl, H.-P. Fortmeyer, W. Marz, H. Scharnagl, A. Oldenburg, S. Angermuller, I. Fleming, K. H. Usadel, and R. Busse Chronic selective hypertriglyceridemia impairs endothelium-dependent vasodilatation in rats Cardiovasc Res, June 1, 1999; 42(3): 783 - 793. [Abstract] [Full Text] [PDF]
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R. Wever, P. Boer, M. Hijmering, E. Stroes, M. Verhaar, J. Kastelein, K. Versluis, F. Lagerwerf, H. van Rijn, H. Koomans, et al. Nitric Oxide Production Is Reduced in Patients With Chronic Renal Failure Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1168 - 1172. [Abstract] [Full Text] [PDF]
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S. A. Gupte, T. Rupawalla, K. M. Mohazzab-H., and M. S. Wolin Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1535 - H1542. [Abstract] [Full Text] [PDF]
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S. R. Thom, D. Fisher, Y. A. Xu, S. Garner, and H. Ischiropoulos Role of nitric oxide-derived oxidants in vascular injury from carbon monoxide in the rat Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H984 - H992. [Abstract] [Full Text] [PDF]
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T. Heitzer, S. Y. Herttuala, E. Wild, J. Luoma, and H. Drexler Effect of vitamin E on endothelial vasodilator function in patients with hypercholesterolemia, chronic smoking or both J. Am. Coll. Cardiol., February 1, 1999; 33(2): 499 - 505. [Abstract] [Full Text] [PDF]
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I. J. Kullo, R. D. Simari, and R. S. Schwartz Vascular Gene Transfer : From Bench to Bedside Arterioscler Thromb Vasc Biol, February 1, 1999; 19(2): 196 - 207. [Full Text] [PDF]
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W. H. Kaesemeyer, R. B. Caldwell, J. Huang, and R. W. Caldwell Pravastatin sodium activates endothelial nitric oxide synthase independent of its cholesterol-lowering actions J. Am. Coll. Cardiol., January 1, 1999; 33(1): 234 - 241. [Abstract] [Full Text] [PDF]
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T. Marumo, V. B. Schini-Kerth, R. P. Brandes, and R. Busse Glucocorticoids Inhibit Superoxide Anion Production and p22 Phox mRNA Expression in Human Aortic Smooth Muscle Cells Hypertension, December 1, 1998; 32(6): 1083 - 1088. [Abstract] [Full Text] [PDF]
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Y. Xia, A.-L. Tsai, V. Berka, and J. L. Zweier Superoxide Generation from Endothelial Nitric-oxide Synthase. A Ca2+/CALMODULIN-DEPENDENT AND TETRAHYDROBIOPTERIN REGULATORY PROCESS J. Biol. Chem., October 2, 1998; 273(40): 25804 - 25808. [Abstract] [Full Text] [PDF]
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