Vol. 283, Issue 2, H750-H759, August 2002
Native LDL and minimally oxidized LDL differentially regulate
superoxide anion in vascular endothelium in situ
David W.
Stepp1,
Jingsong
Ou2,4,
Allan W.
Ackerman2,
Scott
Welak2,6,
David
Klick3, and
Kirkwood A.
Pritchard Jr.2,3,4,5
Departments of 1 Physiology,
2 Surgery (Division of Pediatric Surgery),
3 Pharmacology and Toxicology,
4 Cardiovascular Center, 5 Free Radical
Research Center, and 6 Summer Practicum in
Undergraduate Research Studies Medical College of Wisconsin, Milwaukee,
Wisconsin 53226
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ABSTRACT |
Low-density
lipoprotein (LDL) and its oxidized derivatives are hypothesized to
impair vascular function by increasing superoxide anion
(O
·). To investigate mechanisms in situ, isolated
carotid arteries were incubated with native LDL (nLDL) or minimally
oxidized LDL (mmLDL). With the use of en face fluorescent confocal
microscopy and hydroethidine, an oxidant-sensitive fluorescent probe,
we found that nLDL increased O· in vascular
endothelium greater than fourfold by an
N
-nitro-L-arginine methyl ester
(L-NAME)-inhibitable mechanism. In contrast, mmLDL
increased O· in vascular endothelium greater than
eightfold by mechanisms that were partially inhibited by
L-NAME and allopurinol and essentially ablated by
diphenyleneiodium. These data indicate that both nLDL and mmLDL
uncouple endothelial nitric oxide synthase (eNOS) activity and that
mmLDL also activates xanthine oxidase and NADPH oxidoreductase to
induce greater increases in O· generation than
nLDL. Western analysis revealed that both lipoproteins inhibited
A-23187-stimulated association of heat shock protein 90 (HSP90) with
eNOS without inhibiting phosphorylation of eNOS at serine-1179
(phospho-eNOS), an immunological index of electron flow through the
enzyme. As HSP90 mediates the balance of ·NO and
O· generation by eNOS, these data provide new
insight into the mechanisms by which oxidative stress, induced by nLDL
and mmLDL, uncouple eNOS activity to increase endothelial
O· generation.
native low-density lipoprotein; minimally oxidized low-density
lipoprotein; smooth muscle cells; superoxide anion; confocal
microscopy; hydroethidine
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INTRODUCTION |
ONE MECHANISM by
which hypercholesterolemia is believed to induce vascular dysfunction
is by increasing O· generation. Although ample,
indirect evidence exists to suggest that hypercholesterolemia increases
O· production by the vessel wall (16, 17,
22), the cells involved and the mechanisms by which native
low-density lipoprotein (nLDL) and its oxidized derivatives alter
vascular O· production remain unclear. For
example, hypercholesterolemia increases adherence of mononuclear cells
to the vessel wall (19, 29). Recruitment and subsequent
migration of mononuclear cells into the vessel wall introduces new
cellular sources of O·. Even before recruitment,
high plasma concentrations of LDL may promote hyperresponsiveness of
circulating blood elements (12). Thus mononuclear cells
entering the vessel wall may be predisposed to generate increased
quantities of O· after transmigration.
Hypercholesterolemia also increases nLDL entry into the vessel wall,
where it becomes trapped and subsequently oxidized (20, 21). Once oxidized it is capable of inducing a wide variety of
potent proinflammatory responses in the vasculature leading to impair
function. Many studies have shown that the effects of LDL on
endothelial and vascular function depend strongly on the degree of
oxidation. For example, one essential function of the endothelium is to
limit the accumulation of lipid hydroperoxides (LOOH) in nLDL
(26). When the content of LOOH in LDL exceeds a critical
threshold, endothelial cells begin to participate in the oxidation
process (26). In addition to endothelial cells, smooth
muscle cells can also oxidize LDL by a
O·-dependent process (13).
Whereas many studies have implicated O· as a key
factor in the pathogenesis of atherosclerosis, the direct effects of
LDL on vascular endothelial O· production have not
been systematically examined. Canines for the most part are resistant
to diet-induced atherosclerosis, although some colonies have been
established that are sensitive to cholesterol if fed for an extended
time (4-8 mo) (1). Recent studies found that
inhibiting platelet function significantly reduced the progression but
did not eliminate atherosclerosis (1). These observations suggest that high plasma concentrations of cholesterol are able to
induce atherogenesis in the canine.
The goal of this study was to test the hypothesis that lipoproteins, in
the absence of other confounding influences, increase O· generation by vascular endothelium in situ.
Previously, we and colleagues (25) and others
(32) incubated cultured endothelial cells with nLDL and/or
oxidized LDL and found that these lipoproteins increased
O· production by uncoupling endothelial nitric
oxide synthase (eNOS) activity as an initial step in sorting out the
mechanisms involved. Although both nLDL and oxidized LDL increased
O· generation in cultured endothelial cells, it is
unknown whether they would increase O· in situ,
and, if so, by what cell type and oxidative enzyme. Answering these questions required an alternative analytic approach for assessing O· generation in vascular tissues.
In this investigation, we incubated isolated carotid arteries with
purified nLDL and minimally oxidized LDL (mmLDL) to determine their
effects on vascular O· generation. We used
fluorescent confocal microscopy and hydroethidine (HEt), an
oxidant-sensitive, intracellular fluorescent probe, to identify which
cells were generating O·. Conversion of HEt to
ethidium (Et) was examined with respect to radical specificity. Sources
of oxidative enzyme activity were determined with specific and
selective inhibitors. Finally, as nLDL and oxidized LDL were reported
to uncouple eNOS activity, the effects of these atherogenic lipoproteins on eNOS in cultured endothelial cells were examined by
Western blot analysis for changes in phospho-eNOS (serine-1179) and
association of heat shock protein 90 (HSP90).
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MATERIALS AND METHODS |
Preparation of nLDL.
nLDL was isolated by sequential density ultracentrifugation
(1.019-1.063 g/ml) from pooled human plasma (2-5 donors per
isolation) as previously described (25). Butylated
hydroxytoluene, a lipid-soluble antioxidant, and EDTA were added
immediately to the plasma (final concentrations = 20 µM and
0.01%, respectively). The plasma was mixed for 15 min at room
temperature before ultracentrifugation. Sterile techniques, reagents,
and dialysis solutions were used for all isolation steps. Cholesterol
concentrations were determined using the cholesterol oxidase kit from
Sigma (catalogue number 352-20). nLDL was used within 1 wk of isolation.
Preparation of mmLDL.
nLDL was isolated as outlined above. The LDL was placed in a sterile
50-ml conical tube and stored at 4°C for at least 3-6 mo before
use for experiments as described (4, 8). mmLDL had a
significantly higher content of LOOH than nLDL as measured by the
tri-iodide assay (mmLDL = 73.8 ± 25.0 vs. nLDL = 0.3 ± 5.0 nmol LOOH/mg cholesterol, P < 0.01, n = 5) (26).
Tissue extraction.
Canine carotid arteries were obtained from adult mongrel dogs of either
gender. Vessels were quickly excised and transferred to physiological
saline solution at room temperature, and surrounding advential tissue
was quickly cleaned away. The artery was sectioned into segments of at
least 2 cm long and placed in RPMI 1640 containing 10% fetal bovine
serum (FBS), 20 mM HEPES, and antibiotics-antimycotics (termed RPMI
medium hereafter). After being washed twice with RMPI 1640 to remove
adherent blood elements, the vessels were placed into organ culture.
Organ culture.
Canine carotid artery segments were placed in a 60-mm dish containing
either control RPMI medium or RPMI medium containing nLDL (80, 160, or
240 mg/dl) or mmLDL (240 mg/dl). The arterial segments were incubated
at 37°C, 5% CO2-95% air and 100% H2O for 24 h.
Superoxide anion detection.
Radical specificity of HEt conversion to Et was examined in phosphate
buffer (250 mM, pH 7.45) containing HEt (10 µM). Fluorescence was
monitored with respect to time on a Perkin Elmer LS50B (excitation wavelength 488 nm, emission wavelength 620 nm, 10-nm slit
widths). Superoxide anion was generated with xanthine/xanthine
oxidase (30, 31). First, xanthine was added (final
concentration = 1 mM) and mixed, and fluorescence was recorded.
Xanthine oxidase was then added (final concentration = 14 U/ml),
the solution mixed, and fluorescence recorded. Finally, superoxide
dismutase (SOD) was added (final concentration = 1,000 U/ml) to
inhibit O·-dependent increases in Et fluorescence.
The solution was mixed and fluorescence recorded. The effect of Tiron,
a well-known cell-permeable, O·-dismutating agent
(34) was determined in a separate incubation by
substituting Tiron (final concentration, 10 mM) for SOD.
To determine the effects of ·NO on O·-dependent
increases in Et fluorescence
2-(N,N-diethylamino)-diazenolate-2-oxide sodium
salt (DEA NONOate, final concentration = 14 µM) was added to
cuvettes containing HEt (10 µM), xanthine (1 mM), and xanthine oxidase (14 U/ml), and the solution was mixed, and fluorescence was
recorded. As ·NO reacts with O· to form
peroxynitrite, the effect of synthetic peroxynitrite on conversion of
HEt to Et was also determined. Freshly prepared peroxynitrite was added
as a single bolus (final concentration = 200 µM) to the HEt
phosphate reagent with and without glutathione (3 mM), the solution was
mixed, and fluorescence was recorded.
Manganese-5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP) is a
cell-permeable SOD mimetic, which has been shown to protect against
paraquat-induced lung injury (7). Although MnTBAP is well
recognized as a SOD mimetic, it is also a metal heme compound that may
quench fluorescence as well as dismutate O·. To
investigate this possibility we assessed changes in fluorescence in
Et-labeled endothelial cells in the presence and absence of MnTBAP.
Endothelial cells were incubated in Hanks' balanced salt solution
(HBSS) containing A-23187 (5 µM) and HEt (10 µM) for 30 min. The
cells were removed from the wells with trypsin, and the median
fluorescence per cell was determined by FLOW analysis. MnTBAP (10 µM)
was added to the suspension of Et-labeled cells and incubated for 10 min. The median fluorescence/cell in the MnTBAP-treated, Et-labeled
cell suspension was determined by FLOW analysis. Finally, the cells
were washed free of MnTBAP by centrifugation and resuspended in HBSS,
and the median fluorescence per cell was determined.
To determine lipoprotein-induced changes in vascular
O· generation control, nLDL, and mmLDL arterial segments were placed in 5 ml of HBSS-containing HEt (10 µM) for 30 min at room temperature. After incubation, the segments were washed
free of HEt with three 10-min washes with HBSS and then placed on a
coverslip that was moistened with physiological saline solution (PSS),
endothelial side down. The PSS used in these experiments was
equilibrated with 21% O2-5% CO2-74%
N2 and had the following composition (mM): 119 NaCl, 4.7 KCl, 1.17 MgSO4, 1.6 CaCl2, 1.18 NaH2PO4, 24 NaHCO3, 0.026 EDTA, and
5.5 glucose. Quantification of the intensity of Et fluorescence in
arterial tissues was achieved by analysis of images using NIH Image
version 1.62. Four to six regions that contained no evidence of
staining were used to calculate the average background fluorescence for
each image. The fluorescence intensity in 12 cells was then measured,
and the mean fluorescence intensity was calculated and corrected for
background fluorescence. Data are presented as means ± SE in
arbitrary units and were analyzed by analysis of variance (ANOVA) of
paired samples with Fisher's paired least-significant difference test
as a post hoc test. Black and white images were pseudocolorized to more
clearly illustrate the magnitude of changes in intensity of Et staining.
Inhibitor studies.
To determine the effects of nLDL and mmLDL on
non-O·-dependent conversion of HEt into Et,
arterial segments were preincubated with MnTBAP (10 µM) for 30 min,
and Et fluorescence in the isolated arterial segment was recorded by
confocal microscopy. Accordingly, the images obtained from control,
nLDL, and mmLDL pretreated with MnTBAP represented the extent to which
non-O·-dependent mechanisms converted HEt to Et.
The contribution of O· from different oxidative
enzymes in the isolated arterial segments was determined by
preincubating matched arterial segments with L-NAME (500 µM), allopurinol (100 µM), or diphenyleneiodonium (DPI, 100 µM)
30 min before and during incubation in PSS containing HEt (10 µM,
final concentration). The rationale for using these inhibitors is as
follows. L-NAME is a substrate analog inhibitor of eNOS, which inhibits the generation of ·NO and O· (18, 25). Allopurinol is a competitive inhibitor of
xanthine oxidase that has been shown to inhibit O· generation from xanthine oxidase bound to the endothelium of aortas from hypercholesterolemic rabbits (33). DPI prevents NADPH
from binding to flavoproteins, resulting in inhibition of all
flavinoid-dependent enzyme activity (23, 31) including NOS
isozymes (27, 31).
Effects of nLDL and mmLDL on the association of HSP90 with eNOS.
Bovine aortic endothelial cells (BAEC) were incubated with nLDL (240 mg/dl) or mmLDL (240 mg/dl) in RPMI medium for 24 h. After
incubation, the cultures were washed three times with HBSS and then
incubated in HBSS containing L-arginine (10 µM) with and
without A-23187 (5 µM) for 10 min. The HBSS buffer was removed by
aspiration, and cell proteins were harvested in modified RIPA buffer
containing 1:100 dilution of phosphatase inhibitor cocktail I (P2850,
Sigma Chemical; St. Louis, MO) and other inhibitors as described
(24). To determine the effects of nLDL and mmLDL on eNOS
activation, eNOS was immunoprecipitated from lysates of the cultures
using established protocols (24). Coprecipitated proteins
were separated by SDS-PAGE (7.5%), transferred to nitrocellulose, and
blotted for phospho-eNOS (serine-1177/serine-1179), eNOS, and HSP90 as
described (24).
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RESULTS |
Detection of O
· generation.
A cell-free system was used to determine the specificity of reactive
oxygen species conversion of HEt to Et. Some of the reagents in these
experiments were dissolved in NaOH to increase their stability. To
ensure that reactions took place in constant pH, the HEt was dissolved
in 250 mM phosphate buffer (pH 7.45). Xanthine had little effect on the
baseline levels of Et fluorescence (Fig. 1). Upon addition of xanthine oxidase,
however, marked increases in Et fluorescence were observed (rate = 0.0458 ± 0.005 U/s) that were completely blocked by SOD. Previous
cell-free studies indicated that hydrogen peroxide (200 µM) increased
conversion of HEt to Et to ~10% of the fluorescence induced by
potassium superoxide (200 µM) (5). If hydrogen peroxide
contributed significantly to the conversion of HEt to Et under these
conditions, then SOD should have partially reduced this rate rather
completely reducing it to zero. Accordingly, we interpret these data to
mean that hydrogen peroxide formed from dismutation of
O· contributes little to the conversion of HEt to
Et.
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Fig. 1.
Conversion of hydroethidine (HEt) to ethidine (Et).
Increases in fluorescence due to xanthine (X)/xanthine oxidase (XO)
generation of superoxide anion are shown. Freshly prepared solutions of
HEt (10 µM) in phosphate buffer (250 mM, pH 7.45) possess low levels
of fluorescence in the presence of xanthine alone. Addition of XO to
the cuvette causes a marked increase conversion of HEt to Et, which is
highly fluorescent. Addition of superoxide dismutase (SOD, 1,000 U/ml)
blocks further increases, whereas addition of
2-(N,N-diethylamino)-diazenolate-2-oxide sodium
salt (DEA NONOate, 14 µM) attenuates the rate of increase in Et
fluorescence. These line graphs show that the conversion of HEt to Et
is superoxide anion dependent.
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Another way of preventing O· from reacting with
HEt is by scavenging it with ·NO before it has a chance to react with
the fluorescent indicator (14). When DEA NONOate was added
to the cuvette as a source of ·NO, the rate of increase in Et
fluorescence was markedly reduced (rates pre-DEA NONOate = 0.0458 ± 0.005 vs. post-DEA NONOate = 0.007 ± 0.003 U/s). The ·NO-attenuated rate represented ~15% of the rate before ·NO addition. The effects of ·NO from DEA NONOate appeared to be
dose dependent, in that lower concentrations yielded smaller attenuations in the rate of increase in Et fluorescence (data not
shown). Such findings are in agreement with others showing that the
balance of ·NO and O· generation plays an
important role in mediating conversion of oxidant-sensitive fluorescent
probes (14).
As ·NO reacts with O· to form peroxynitrite,
which is a potent oxidant that is reported to convert HEt to Et
(28), the effects of peroxynitrite on HEt conversion were
also examined. The addition of freshly synthesized peroxynitrite as a
single bolus (200 µM, final concentration) to the HEt-phosphate reagent increased baseline levels of Et fluorescence from 0.189 ± 0.010 to 0.699 ± 0.020 units. Although this increase in Et
fluorescence was 2.4 times the baseline, it represented a small portion
of the Et fluorescence that was achieved when O· was generated with xanthine/xanthine oxidase (~10%). More
importantly, glutathione markedly inhibited peroxynitrite-mediated
conversion of HEt to Et (0.189 ± 0.010 to 0.213 ± 0.010 units). These data show that in the presence of physiologically
relevant concentrations of glutathione, peroxynitrite induces minimal
increases in Et fluorescence. In contrast, O·
readily converted HEt to Et in the presence of glutathione (0.046 ± 0.006 U/s). Accordingly, we interpret these data to mean that under the physiological conditions that occur in vascular cells or isolated arterial segments, peroxynitrite more likely reacts with glutathione than with HEt.
With nearly all assays for O·, specificity is
afforded by SOD. The extent to which SOD reduces analytic signals is
considered an acceptable indirect measure of O·
production. The problem with this approach when measuring intracellular
levels of O· is SOD is impermeable to cells. To
circumvent this problem, we incubated arterial segments with MnTBAP, a
well-recognized, cell-permeable SOD mimetic (7). Zuo et
al. (35) found that MnTBAP reduced Et fluorescence in
arterial segments to levels that were similar to those reported for
Tiron. Using MnTBAP to reduce O·-dependent increases in Et fluorescence, the effects of nLDL and mmLDL on non-O·-dependent increases in Et fluorescence could be easily evaluated.
Because MnTBAP is a metal heme compound, it also may reduce
fluorescence by quench (15) rather than by scavenging
O·. To determine whether quench was involved in
the mechanisms by which MnTBAP reduced Et fluorescence, we added MnTBAP
to endothelial cells that were previously incubated with HEt. The
median fluorescence per cell in the Et-stained endothelial cells was
1,229 ± 4 (n = 3). After incubation with MnTBAP,
the median fluorescence per cell in the MnTBAP-treated, Et-stained
cells was 1,240 ± 10 (n = 3). After MnTBAP was
removed by centrifugation, the median fluorescence per cell was
1,235 ± 10 (n = 3). These data show that the
median fluorescence per cell was essentially unchanged by MnTBAP. On the basis of these findings, we conclude that MnTBAP does not quench
preexisting Et fluorescence in vascular endothelium. With this
information as background, we proceeded to investigate the mechanisms
by which nLDL and mmLDL altered vascular O· generation in situ.
Arterial segments were analyzed for patterns of Et staining by en face
fluorescent confocal microscopy and image analysis. One of the benefits
of confocal microscopy is that thick samples or tissues sections can be
optically dissected. The endothelial layer was identified by first
focusing above the plane of endothelium at the edge of the cut vessel,
then in the plane of the endothelium, and finally below the plane of
the endothelium (Fig. 2, A,
B, and C, respectively). The images captured
above the plane of the endothelium showed small bubbles trapped between
the endothelium and the glass slide and marked increases in Et staining
in the region where the vessel was cut (Fig. 2, A, bright
section in the top left corner). Moving toward the center of the
arterial segment and adjusting the focal plane deeper into the en face preparation revealed an intact endothelial cell monolayer (Fig. 2B). Endothelial cells were elongated and oriented in
direction of blood flow in the vessel. Focusing deeper into the
arterial tissue below the plane of the endothelium revealed a layer of vascular smooth muscle cells (Fig. 2C). Smooth muscle cells
were elongated and oriented perpendicular to blood flow. These black and white images are from a vessel that was incubated with mmLDL, which
markedly increased Et staining in the endothelium and often in the
smooth muscle cells and represent typical data obtained by en face
confocal microscopy. For presentation purposes, images were cut and
pasted into the same file and pseudocolorized. Figure 2D is
a pseudocolorized image of a carotid artery incubated with nLDL. Figure
2E is a pseudocolorized image of a carotid artery incubated
with nLDL and MnTBAP, suggesting that the increase in Et fluorescence
induced in Fig. 2D was O· dependent.
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Fig. 2.
En face confocal fluorescent microscopy of superoxide
anion production in situ. Representative black and white images of en
face fluorescent confocal micrographs of canine carotid arteries are
shown. A: fluorescence at the edge of a cut arterial
segment. B: fluorescence in the endothelium of an arterial
segment incubated for 24 h with minimally oxidized low-density
lipoprotein (mmLDL, 240 mg/dl). C: fluorescence in the
smooth muscle cell layer of the same arterial segment as in
B. D: pseudocolorized image of endothelium
from enface fluorescent confocal micrograph of an arterial
segment incubated for 24 h with native LDL (nLDL, 240 mg/dl).
E: pseudocolorized image of endothelium from arterial
segment incubated for 24 h with nLDL (240 mg/dl) pretreated with
manganese-5,10,15,20-tetrakis(4-benzoic acid)porphyrin for 30 min
before incubation with HEt.
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nLDL increases vascular O·
generation.
nLDL significantly increased vascular O·
production in a concentration-dependent fashion (Fig. 3A). When arterial segments
were incubated with a single concentration of nLDL (240 mg/dl),
O·-dependent Et fluorescence was significantly
increased at 6 h and again at 24 h (Fig. 3B).
L-NAME significantly inhibited nLDL-induced increases in
O· (P < 0.01), suggesting that eNOS was a major source of O· in nLDL-treated arterial segments (Fig. 4). The role of
xanthine oxidase and NADPH oxidoreductase in the nLDL-induced increase
in O· production was not assessed. The rationale
for this decision lies in the observation that L-NAME
reduced O· generation by ~64% (Fig. 4). The
remaining 36% of the O· was too small to dissect
out the role of other oxidative enzymes pharmacologically within the
statistical and discriminatory power of the HEt assay. Whereas we
cannot discount a modest role for either xanthine oxidase or NADPH
oxidoreductase in the production of O· in response
to nLDL, it is clear that the majority of O·
generated in the endothelium of arterial segments treated with nLDL
appears to originate from eNOS. It should be noted that
L-NAME reduced Et staining in the nLDL-treated arterial
segments to essentially the same extent as DPI did in the mmLDL-treated
arterial segments.
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Fig. 3.
nLDL increases endothelial superoxide anion production in situ.
Relative changes in superoxide anion, as assessed by fluorescence
intensity of Et, in the endothelium of arterial segments incubated with
nLDL (240 mg/dl) with respect to concentration (A) and time
(B) are shown. Data represent means ± SE. Statistical
analysis was two-tailed Student's t-test with Welch's
correction (**P < 0.01, n = 5). NS,
not significant.
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Fig. 4.
nLDL and mmLDL differentially increase endothelial
superoxide anion production in situ via different oxidative enzymes.
Relative changes in superoxide anion, as assessed by fluorescence
intensity of Et, in the endothelium of arterial segments incubated with
nLDL (240 mg/dl, 24 h) and mmLDL (240 mg/dl, 24 h) in the
absence and presence of inhibitors are shown. Data represent means ± SE. Statistical analysis was two-tailed Student's t-test
with Welch's correction (*P < 0.01;
**P < 0.001). nLDL- and mmLDL-treated arterial
segments were incubated with
N-nitro-L-arginine methyl ester
(L-NAME, 500 µM), allopurinol (AL, 100 µM), or
diphenyleneiodonium (DPI, 100 µM) for 15 min before and during
incubation with HEt.
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mLDL increases vascular O·
generation.
Incubation of arterial segments with mmLDL (240 mg/dl)
significantly increased O· in vascular endothelium
greater than two times the levels induced by nLDL and greater than
eight times the levels at baseline (P < 0.01, Fig. 4).
Optical dissection of the mmLDL-treated arterial segments revealed that
most of the increase in Et fluorescence resided in the endothelium with
minor and occasional involvement of smooth muscle cells (data not
shown). This larger increase in O· production made
it possible to examine the role of other oxidative enzymes with greater
confidence. L-NAME significantly decreased Et fluorescence
induced by mmLDL (P < 0.01) confirming observations that oxidized LDL uncouples eNOS activity (32).
Interestingly, L-NAME reduced Et fluorescence in the
mmLDL-treated segments to essentially the same extent as it did in the
nLDL-treated arterial segments (delta change with L-NAME,
mmLDL = 1,900 vs. nLDL = 2,172 arbitrary fluorescent units).
Allopurinol and DPI significantly reduced Et fluorescence in
mmLDL-treated segments by ~56% and ~80%, respectively. Thus mmLDL
increases vascular O· generation by uncoupling
eNOS and activating additional oxidative enzymes xanthine oxidase and
NAD(P)H oxidoreductase. Representative pseudocolorized images of Et
fluorescence in the vascular endothelium of arterial segments
illustrate the prooxidant effects of nLDL and mmLDL and the effects of
selective inhibitors on blocking O· generation
from the different oxidative enzymes (Fig. 4).
Effects of nLDL and mmLDL on the activation state of eNOS.
Typically, control cultures contain low levels of phospho-eNOS and low
levels of HSP90 associated with eNOS. Upon stimulation, control
cultures display marked increases in phosphorylation of eNOS at
serine-1179 and association of HSP90 with eNOS (24). In
Fig. 5, we see the typical pattern of
phospho-eNOS and HSP90 association with eNOS in the control cells. In
this blot, neither nLDL nor mmLDL appeared to alter basal or
A-23187-stimulated levels of phospho-eNOS. These lipoproteins, however,
did alter the association of HSP90 with eNOS. nLDL increased the
association of HSP90 with eNOS under basal conditions, suggesting that
low levels of oxidative stress activate cultured endothelial cells. In
contrast, in cells incubated with mmLDL, the association of HSP90 with
eNOS remained low. When the lipoprotein-treated endothelial cell
cultures were stimulated with A-23187, the association of HSP90 with
eNOS decreased to baseline levels in cells incubated with nLDL (Fig.
5A, lane 5) but failed to increase over control
levels in cells treated with mmLDL (Fig. 5A, lane
4 vs. lane 6). Figure 5B illustrates the
reproducibility of the effects of nLDL and mmLDL on phospho-eNOS, eNOS,
and association of HSP90 in endothelial cultures exposed to nLDL and
mmLDL for 24 h.
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Fig. 5.
nLDL and mmLDL differentially modulate the activation
state of enothelial nitric oxide synthase (eNOS). A: Western
blots of coprecipitated proteins in an immunoprecipitation of eNOS from
bovine aortic endothelial cell cultures incubated for 24 h with
nLDL (240 mg/dl) and mmLDL (240 mg/dl). IP, immunoprecipitation; IB,
immunoblot. Low levels of phospho-eNOS (p-eNOS) are observed in the IP
of eNOS under basal conditions. A-23187 (5 µM) stimulation markedly
increased phospho-eNOS levels, indicating an increase in electron flow
through the enzyme. IB for eNOS shows that the level of eNOS in each
lane is essentially equal. IB for heat shock protein 90 (HSP90) reveals
the nLDL and mmLDL differentially modulate the association of HSP90
with eNOS. Increases in phospho-eNOS without sufficient HSP90 suggest
that nLDL and mmLDL increase eNOS-dependent superoxide anion production
by disrupting normal HSP90 and eNOS interactions. B:
means ± SE for the relative densities for phospho-eNOS/eNOS and
HSP90/eNOS under the treatment conditions indicated as pluses and
minuses in A. Open bars, ratio of a control value of 1.0;
solid bars, endothelial cell cultures treated with nLDL or mmLDL under
basal (nonstimulated conditions); hatched bars, control, nLDL, and
mmLDL cultures stimulated with A-23187.
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DISCUSSION |
In the present study, we used HEt to detect relative changes in
vascular O· production in situ. With this
oxidant-sensitive probe, we were able to identify which cells in the
vessel wall generated O· and obtain
semiquantitative data from the images using conservative analysis
protocols. Our findings indicate that nLDL and mmLDL induced different
oxidative enzymes to increase O· generation in the
endothelium of arterial segments. En face optical dissection of the
vessel wall revealed that nLDL increased O· by an
eNOS-dependent mechanism. mmLDL increased O· primarily in the endothelium, although some vessels showed clear evidence of O· production in the smooth muscle
cells. mmLDL uncoupled eNOS activity as well as increased O· generation from xanthine oxidase and NAD(P)H
oxidoreductase. Such differences reinforce the notion that vascular
disease develops from a series of distinct mechanisms that may or may
not overlap, depending on the extent of oxidative modification of the
LDL particle. These in situ data confirm previous findings by us and
colleagues (25) and Malinski and associates (32) using cultured endothelial cells. Furthermore, our
observations that A-23187 stimulation decreased the association of
HSP90 with eNOS in nLDL-treated endothelial cells and failed to
increase in mmLDL-treated cells, rather than the typical response of
increased association, provide new insight into the cellular mechanisms governing uncoupled eNOS activity.
In recent years HEt has gained popularity as an O·
probe because it is sensitive and easy to use, despite the possibility
it may actually underestimate production (3). Arnal and
associates (2) reported that for measurements of
extracellular O·, SOD-inhibitable increases in Et
fluorescence detected similar variations in O· production as ferricytochrome c and lucigenin. Recent
studies combining confocal microscopy with HEt successfully detected
intracellular O· in the myocytes of heat-stressed diaphragms from mice (35). Here, we used a similar
approach to identify which cells were generating
O· in the vessel wall and specific enzyme
inhibitors to determine which oxidative enzymes were induced after
exposure to nLDL and mmLDL. Identifying which cells were generating
O· was made easier by the fact Et partitions in
the nuclei after conversion. Such partitioning makes the fluorescent
probe less available to reducing agents or reagents that quench. For
example, MnTBAP, which is a metal heme compound that may quench, had no effect on the median fluorescence per cell in endothelial cells prelabeled with Et. Thus, when cells or tissues are incubated with
MnTBAP or Tiron, another O· scavenger that is
accessible to intracellular compartments (35), one can conclude that the observed decreases in Et fluorescence are more likely
due to O· scavenging than quench.
The cell-free studies using SOD indicate that the conversion of HEt to
Et is relatively specific for O·. We found that
when ·NO scavenged O·, the conversion of HEt to
Et was attenuated in a concentration-dependent fashion by >90%.
Freshly synthesized peroxynitrite did not significantly promote HEt
conversion to Et, especially in the presence of glutathione. In
vascular tissues, MnTBAP (a cell-permeable SOD mimetic) reduced Et
fluorescence in the endothelium of isolated arterial segments to
background levels. Our observation that MnTBAP reduced
O·-dependent increases in Et fluorescence was
consistent with findings obtained with Tiron (35). The
decrease in Et fluorescence in the nLDL- and mmLDL-treated arteries
mediated by L-NAME suggests that both atherogenic
lipoproteins induced eNOS to generate O·. The
decreases in Et fluorescence in mmLDL-treated arteries mediated by
allopurinol and DPI indicate that mmLDL increased
O· generation by a xanthine oxidase- and a NADPH
oxidoreductase-dependent mechanism. These conclusions are based on the
fact that these inhibitors are well recognized for blocking
O· generation from their targeted enzymes in other
systems (23, 31, 33). Although conversion of HEt to Et may
underestimate actual O· production
(3), relative increases in endothelial generation of
O· can be assessed with sufficient precision in
situ to allow for statistical discrimination among test groups.
With appropriate protocols, en face fluorescent confocal microscopy
provided precise and reproducible measurements of Et fluorescence in
vascular tissues. After the endothelium was located by adjusting the
microscope through different focal planes, the surface of the lumen was
quickly assessed for endothelial integrity and fluorescent intensity.
Moistening coverslips with PSS before placing arterial segments
endothelial side down helped maintain the integrity of the endothelium.
Marked increases in Et fluorescence were noted in the blood vessels in
regions of the lumen that were damaged due to cutting carotid arteries
into segments. For our analyses, we cut the arteries into segments 2 cm
in length before incubation. This step increased the surface area of
endothelium available for analysis, making it possible to exclude
damaged regions. En face fluorescence microscopy revealed that the
arterial segments also possessed low levels of nonspecific
fluorescence. Accordingly, background measurements of fluorescence in
regions between nuclei were obtained and subtracted from Et
fluorescence in the nuclei of the vascular endothelium. By avoiding
damaged regions and correcting nuclear pixel intensity for nonspecific
fluorescence, we were able to obtain fluorescent intensity data that
revealed that nLDL increased O· by an
endothelium-, eNOS-dependent mechanism and that mmLDL increased
O· predominantly by an endothelium- (with some
involvement of vascular smooth muscle cells), eNOS-, xanthine oxidase-,
and NAD(P)H oxidoreductase-dependent mechanism.
The mechanisms by which LDL induces vascular dysfunction are complex.
Previously, our laboratory focused on mechanisms of endothelial
dysfunction induced by nLDL. When LDL crosses the endothelium and
becomes trapped in the vessel wall, it can be oxidized to a
proinflammatory LDL particle by endothelial cells, smooth muscle cells
and monocytes (4, 6, 13). In this way, multiple mechanisms
can be induced in the vessel wall by the initial insult of increased
plasma concentrations of nLDL. Our findings suggest that the
association of HSP90 with eNOS, which is required for ·NO production
and activity (9, 10), might also be regulated by oxidative
stress. The evidence supporting this statement comes from the fact that
mmLDL contained significantly higher levels of LOOH than nLDL before
incubation. Such changes in oxidation are similar to the levels in
mmLDL reported earlier (8).
The finding that the amount of Et fluorescence inhibited by
L-NAME in mmLDL-treated arterial segments was essentially
equal to that inhibited in the nLDL-treated segments suggests that
these atherogenic lipoproteins uncoupled eNOS to the same extent.
Western blot analysis revealed that the activation state of eNOS was
altered with respect to its association with HSP90. However, neither
lipoprotein significantly altered phospho-eNOS levels under basal or
A-23187-stimulated conditions. nLDL increased the association of HSP90
with eNOS under basal conditions, whereas mmLDL maintained this
association at low levels. In A-23187-stimulated cells, nLDL decreased
the level of HSP90 associated with eNOS compared with the levels in nLDL cells under basal conditions and the levels in A-23187-stimulated control cells. In mmLDL-treated cells, the association of HSP90 with
eNOS failed to increase when stimulated with A-23187. Taken together,
these data indicate that one mechanism may be a decrease in or failure
of HSP90 to associate with eNOS when eNOS is activated. It should be
noted that differences in the regulation of the association of HSP90
with eNOS appeared to be related to the levels of oxidative stress
induced by nLDL and mmLDL. Furthermore, it is important to note that
the lipoprotein with the highest level of oxidative change, mmLDL,
impaired HSP90 interactions with eNOS under both basal and
A-23187-stimulated conditions. Such differences in regulation of this
protein-protein interaction suggest a role for oxidative stress.
Recent findings by Go et al. (11) also indicate that
oxidized LDL uncouples eNOS activity. They report that oxidized LDL induced a nearly threefold increase in phospho-eNOS (serine-1179) levels in BAEC over a period of 30 min. In the initial incubations, citrulline conversion kept pace with phospho-eNOS. However, at a later
time point (30 min), citrulline conversion decreased even though
phospho-eNOS levels remained elevated (11). Our findings here are consistent with those of Go et al. (11) in that a
decrease in citrulline production in the presence of increased levels
of phospho-eNOS also suggests that eNOS generates
O·. Evidence in support of this idea comes from
our data showing that L-NAME decreased Et fluorescence in
mmLDL-treated arterial segments.
In separate studies using diverse redox cycling agents (dichromate VI,
adriamycin, and bis-N-methylacridinium nitrate) to induce
oxidative stress, we found that low levels of oxidative stress appeared
to increase, whereas high levels appeared to decrease HSP90
interactions with eNOS (data not shown). The changes in the association
of HSP90 with eNOS in cells acutely treated with the redox cycling
agents (15 min to 4 h) were similar to the changes induced by
24 h incubation with nLDL representing low levels, and mmLDL,
representing high levels of oxidative stress. Taken together, such
changes indicate that the effects of oxidative stress on the
association of HSP90 with eNOS are both concentration and time dependent.
In summary, nLDL and mmLDL increase vascular endothelial
O· generation in situ. nLDL increases O· by what appears to be an eNOS-dependent mechanism, whereas mmLDL increases O· by eNOS-,
xanthine oxidase- and possibly NAD(P)H oxidoreductase-dependent mechanisms in vascular endothelium of intact arterial segments. Protein-protein interaction studies suggest that the cellular mechanisms by which these atherogenic lipoproteins increase
eNOS-dependent O· generation may involve altering the association of HSP90 with eNOS leading to a change in enzyme function. As increases in O· generation are
believed to play a central role in vascular dysfunction and premature
development of atherosclerosis, therapeutic targeting of these
oxidative pathways to limit O· generation may be a
useful strategy for preserving endothelial and vascular function.
| |
ACKNOWLEDGEMENTS |
The authors thank Jennifer S. Pollock (Vascular Biology Center,
Medical College of Georgia, Augusta, GA) for providing anti-eNOS antibodies and William M. Chilian and Neil Hogg (Medical College of
Wisconsin) for helpful discussions.
| |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-61414 (to K. A. Pritchard, Jr.). D. W. Stepp is the
recipient of an American Heart Association Scientist Development award.
S. Welak was a participant in the Summer Program for Undergraduate Research at the Medical College of Wisconsin working in A. Pritchard's laboratory.
Address for reprint requests and other correspondence:
K. A. Pritchard, Jr., Medical College of Wisconsin, Division
of Pediatric Surgery, Cardiovascular Center, M4066 8701 Watertown Plank
Rd., Milwaukee, WI 53226 (E-mail:
kpritch{at}mcw.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 28, 2002;10.1152/ajpheart.00029.2002
Received 15 January 2002; accepted in final form 18 March 2002.
| |
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ABSTRACT
INTRODUCTION
