From the Department of Biomedical and Surgical Sciences, Verona University, 37134 Verona, Italy and § Department of Bioscience, National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka, 565-8565, Japan
Received for publication, November 26, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Oxidized low density lipoprotein (ox-LDL)
has been suggested to affect endothelium-dependent vascular
tone through a decreased biological activity of endothelium-derived
nitric oxide (NO). Oxidative inactivation of NO is regarded as an
important cause of its decreased biological activity, and in this
context superoxide (O Endothelium-dependent relaxation is impaired in
animals with atherosclerosis (1-3), which has been linked to a
decreased production and/or biological activity of endothelium-derived
nitric oxide (NO)1 (4, 5).
Oxidative inactivation of NO is regarded as an important cause of its
decreased biological activity (6). The vascular release of superoxide
(O Oxidized low density lipoprotein (ox-LDL) has been observed to induce
abnormalities in endothelial function, which may be relevant for the
progression of atherosclerotic lesions (12). In particular functional
alterations of the endothelial cells may be involved in the reduction
of vasodilation, in response to stimuli that induce NO release, in
isolated arteries exposed to ox-LDL (13).
Recently, an endothelial receptor for ox-LDL, called lectin-like ox-LDL
receptor-1 (LOX-1) was cloned from cultured bovine aortic
endothelial cells (BAECs) (14). It has been suggested that ox-LDL
uptake through this receptor may be involved in endothelial activation
or dysfunction in atherogenesis (14). In this context we recently
reported that ox-LDL binding to LOX-1 determined a significant increase
in the generation of reactive oxygen species (ROS) in endothelial cells
(15). In this report we investigated the relationship between
the intracellular production of ROS and in particular of O LDL Isolation--
Whole blood, obtained by venipuncture from
healthy volunteers after 12 h of fasting, was collected into
Vacutainer tubes (Becton Dickinson, Meylan, France) containing
EDTA (1 mg/ml) and processed for LDL separation within 1 day by
sequential flotation in NaBr solution (16) containing 1 mg/ml EDTA.
LDL Oxidation and Modification--
Cu2+-modified
LDL (1.7 mg of protein/ml) was prepared by exposure of LDL to 5 µM CuS04 for 18 h at 37 °C as
described previously (17, 18). The extent of LDL oxidation was
determined by thiobarbituric acid-reactive substances as reported (18).
Protein was measured by the Pierce BCA protein assay reagent (19).
Malondialdehyde-modified LDL (MDA-LDL) was prepared according to a
previously described method (20, 21). Acetylation of LDL was achieved
by repeated additions of acetic anhydride (22).
Cell Cultures--
BAECs were isolated and cultured as described
previously (23). Cells used for experiments were at passage levels
between 2 and 4. Chinese hamster ovary-K1 (CHO-K1) cells and a CHO-K1 cell line stably expressing bovine LOX-1 (BLOX-1-CHO) (14) were cultured as described previously (23). Cell survival was monitored according to the method of Landegren (24).
ROS and O
Confluent BAECs in 24-well plates were incubated in Dulbecco's
modified Eagle's medium (Sigma) containing 10% fetal bovine serum, 10 µM 2',7'-dichlorofluorescin diacetate (Eastman
Kodak Co., Rochester, NY), or 1 µM HE (Kodak) for 20 min.
Increasing concentrations (50-150 µg of protein/ml) of ox-LDL,
native LDL (n-LDL), acetyl-LDL (Ac-LDL), and MDA-LDL were then added to
the medium for 5 min at 37 °C in the presence of 5 mM
arginine and 3 µM tetrahydrobiopterin (TB4). The
incubation time was chosen on the basis of previous data showing that
in these experimental conditions the ROS generation induced by ox-LDL
increased rapidly in the first 5-6 min and then plateaued for longer
ox-LDL incubations (15). Furthermore the short incubation time was
chosen to avoid interferences derived from ox-LDL internalization.
Samples were washed twice with phosphate-buffered saline containing
bovine serum albumin and analyzed with 7000 cells per sample by flow cytometry (Coulter Electronics GmBH, Germany).
To test the response specificity, some radical scavengers such as
vitamin C, trolox, and probucol (at a concentration of 5 µM; Sigma), anti-LOX-1 monoclonal antibody (mAb) (14), or
comparable amounts of nonimmune mouse IgG (14) were incubated with
BAECs, CHO-K1, and BLOX-1-CHO cells under the experimental conditions specified above.
To determine which oxidative systems contribute to the release of
O NO Measurement--
DAF-2 DA is a fluorescent indicator that
enables the direct detection of NO under physiological conditions by
flow cytometry (30). Confluent BAECs in 24-well plates were incubated
in KRP (120 mM NaCl, 4.8 mM KCl, 0.54 mM CaCl2, 1.2 mM MgSO4,
11 mM glucose, 15.9 mM
Na3PO3, pH 7.2) containing 10 µM
DAF-2 DA for 10 min at 37 °C. Cells were then stimulated with 100 nM bradykinin and 150 mM thrombin for 5 min in
presence of 5 mM arginine and 3 µM TB4. To
verify whether the fluorescent signal obtained after the addition of
DAF-2 DA was dependent on the presence of NO, L- and
D-NMMA (200 µM) were preincubated with BAECs
for 30 min before the addition of DAF-2 DA and NO agonists. Samples
were then washed twice with phosphate-buffered saline containing bovine
serum albumin and analyzed with 7000 cells per sample in flow cytometry
(Coulter Electronics GmBH, Germany). In the same experimental
conditions we also evaluated NO production by measuring levels of
nitrite in the cell media by Griess reaction as described previously
(31).
To evaluate the effect of ox-LDL on intracellular NO concentration,
increasing amounts (50-150 µg of protein/ml) of ox-LDL, n-LDL,
Ac-LDL, and MDA-LDL were incubated with BAECs for 0.5-15 min after the
addition of DAF-2 DA and NO agonists, in the presence of 5 mM arginine and 3 µM TB4.
To verify whether the effect of ox-LDL on intracellular NO
concentrations was dependent on ROS production and to test the response
specificity, vitamin C, anti-LOX-1 mAb (14), or comparable amounts of
nonimmune mouse IgG (14) were also used under the experimental
conditions specified above.
Endothelial Nitric Oxide Synthase (eNOS) Activity
Measurement--
The effect of ox-LDL on eNOS metabolism of
3H arginine to 3H citrulline was
determined as described previously (32-34). The assay was performed
under apparent Vmax conditions (32-34). Briefly
BAECs lysates were suspended in cold lysis buffer (0.3 M
sucrose, 10 mM HEPES, 1% Nonidet P-40, 0.1 mM
EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 2 µg/ml
aprotinin, 10 µg/ml soybean trypsin inhibitor, and 50 µM phenylmethylsulfonyl fluoride, pH 7.4) and vortexed. Cell lysates (150 to 250 µg of protein) were combined with NADPH (2 mM), CaCl2 (230 µM), TB4 (3 µM), and 3H-arginine (0.2 µCi, 10 µM) for 20 min at 37 °C. The assay volume was kept
constant at 100 µl. To determine whether ox-LDL altered inducible NOS
activity, the assay was repeated with EDTA (1.7 mM)
replacing calcium in the assay buffers.
Statistical Analysis--
Statistical analysis was performed by
analysis of variance and subsequently by post hoc analysis,
using the SYSTAT program and statistical software manual (SYSTAT Inc.,
Evanston, IL) for Macintosh.
In our experimental conditions the incubations of BAECs with 10 µM DAF-2 DA for 10 min at 37 °C followed by
stimulation with bradykinin or thrombin for 5 min generated a sharp
increase of mean fluorescence intensity (MFI). This increase was
dose-dependently suppressed by the NO synthase inhibitor
L-NMMA whereas D-NMMA, the optical isomer of
L-NMMA, was inactive. Fig. 1
shows the effect of 200 µM L-NMMA and
D-NMMA on basal and stimulated NO production in BAECs.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of L- and
D-NMMA on basal and stimulated intracellular NO
concentration in BAECs. BAECs were preincubated with 200 µM L- and D-NMMA for 30 min at
37 °C before the addition of 10 µM DAF-2 DA for 10 min
(Basal). Cells were then stimulated with 100 nM
bradykinin and 150 mM thrombin for 5 min. Results are
expressed as MFI and are the means ± S.D. of experiments
performed in triplicate on six separate occasions. *, p < 0.001 versus control (no L- or
D-NMMA).
Also the cumulative production of NO as evaluated by measuring levels of nitrite in the media was significantly increased after stimulation of BAECs with bradykinin or thrombin for 10 min at 37 °C (basal = 110 ± 7 pmol/well/h; after bradykinin = 370 ± 14 pmol/well/h, p < 0.001; after thrombin = 410 ± 12 pmol/well/h, p < 0.001).
The exposure of 1.7 mg of protein/ml of n-LDL to 5 µM Cu2+ for 18 at 37 °C resulted in a significant increase of thiobarbituric acid-reactive substances (11.9 ± 1.1 nmol/mg of LDL protein) compared with native LDL (0.24 ± 0.04 nmol/mg of LDL protein; p < 0.001).
The incubation of BAECs with increasing amounts of ox-LDL for 5 min in
the presence of DAF-2 DA, dose-dependently reduced basal
and bradykinin- or thrombin-induced intracellular NO formation (p < 0.001) (Fig. 2)
whereas n-LDL did not (data not shown). Similarly Ac-LDL and MDA-LDL,
even at the highest concentration (200 µg of protein/ml), had no
effect (data not shown). The preincubation of BAECs with 200 µg of
ox-LDL protein also significantly reduced the basal and stimulated
levels of nitrite (basal from 102 ± 6 pmol/well/h to 44 ± 4 pmol/well/h, p < 0.01; after bradykinin from 352 ± 15 pmol/well/h to 121 ± 12 pmol/well/h, p < 0.01; after thrombin from 401 ± 14 pmol/well/h to 184 ± 9 pmol/well/h, p < 0.01). From the evaluation of the
time course of nonstimulated and stimulated BAECs, it is evident that
the effect of ox-LDL on intracellular NO production was already present
after less than 60 s of incubation (Figs.
3, a-c).
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The incubation of BAECs with ox-LDL for 5 min also induced a sharp and
dose-dependent increase in intracellular concentration of
ROS and O
To test the specificity of ROS and O
|
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On the basis of the results described above, to test whether the
reduction of intracellular NO concentration induced by ox-LDL was
dependent on O
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The effect of ox-LDL on eNOS activity was examined by the 3H citrulline assay. ox-LDL did not significantly modify the ability of eNOS to metabolize L-arginine to L-citrulline (native LDL = 64.6 ± 9.4 pmol citrulline/mg protein/min; ox-LDL = 58.7 ± 8.9 pmol citrulline/mg protein/min, p = not significant). In presence of EDTA, the activity of inducible NOS was almost undetectable.
We also analyzed which oxidative systems may contribute to the release
of O
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DISCUSSION |
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Using a novel fluorescence indicator, DAF-2 DA, for direct
detection of NO (30), in this study we examined the relationship between the intracellular production of ROS and in particular of
O
In our experimental conditions the incubations of BAECs with 10 µM DAF-2 DA for 10 min at 37 °C generated an increase in fluorescence intensity both in basal and agonist-stimulated cells, which was dose-dependently suppressed by the NO synthase inhibitor L-NMMA. These results are consistent with several lines of evidences suggesting that NO is generated under basal conditions by endothelial cells (35) and are in agreement with published results showing that 5-min exposure of BAECs to thrombin or bradykinin results in a sharp increase of NO (36). We found that ox-LDL, but not n-LDL or other forms of modified LDL, reduced in a dose-dependent fashion and very rapidly the intracellular NO concentration in basal and stimulated endothelial cells.
Our results agree with a series of studies addressing the effects of ox-LDL on arterial rings (37, 38) and on cultured endothelial cells (39). These effects have been seen to occur when vascular segments or cultured cells are placed in contact with LDL for long periods suggesting inhibition of NO synthesis by ox-LDL. Even if there is no agreement regarding interpretation of this phenomenon (38-40), in our experimental conditions ox-LDL did not significantly alter the ability of eNOS to metabolize L-arginine to L-citrulline. Because the conversion of 3H arginine into 3H citrulline, under apparent Vmax conditions (32-34), is a measure of eNOS levels, the results of this study show that ox-LDL did not alter, at least quantitatively, the ability to produce NO.
Interestingly and in agreement with very recent data
published by our group (15), the results of this study also demonstrate that the rapid decrease in NO induced by ox-LDL was parallelled by a
specular fast increase in ROS and O
The decrease of intracellular NO concentration was
prevented by preincubating BAECs with different antioxidants known to
work as radical scavengers and with anti-LOX-1 mAb. The data support the conclusion that the incubation of ox-LDL with BAECs is associated with a receptor-dependent, abnormally increased
intracellular production of ROS and in particular of O
There are many enzymatic sources for ROS in almost all cell
types (41), and several findings have indicated an increase in ROS upon
receptor ligation (42-46). As for the potential sources of ROS induced
by the binding of ox-LDL to LOX-1, we found that allopurinol and
aspirin did not significantly affect O
Finally the results of this study clearly show that DPI
drastically reduced O
The reduction in intracellular NO concentration as a result of
O
Increased oxidative stress within the vascular wall facilitates
oxidation of LDL (60). The ROS produced by the ligation of ox-LDL to
LOX-1 could facilitate the oxidation of native LDL or partially
oxidized LDL, which in turn could up-regulate LOX-1 expression (61) and
contribute to further O
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FOOTNOTES |
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* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Takeda Science Foundation; and DNO Medical Research Foundation.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.
To whom correspondence should be addressed: Dipartimento di
Scienze Biomediche e Chirurgiche, c/o Medicina D, Ospedale Policlinico, Università di Verona, 37134 Verona, Italy. Tel.: 39-045-8074806; Fax: 39-045-583041; E-mail: comina@medicinad.univr.it.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M010612200
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ABBREVIATIONS |
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The abbreviations used are:
NO, nitric oxide;
O
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