238 Journal
of Atherosclerosis
Original
Articles
and
Vol. 7, No. 4
Thrombosis
Lysophosphatidylcholine Enhances Endothelial NADH/NADPH Oxidase
Saari Takeshita1, Nobutaka Kawashima1, Riichi Tawa2, 1
Inoue1, Hiromu
Dayaun Sakurai2,
First Department of Internal Medicine, Kobe University Department of Analytical and Bioinorganic Chemistry,
2
Superoxide
Anions Production
Gao2, Yoshiyuki and Mitsuhiro
School of Medicine, Kyoto Pharmaceutical
Rikitake1, Yokoyama1
via
Seinosuke
Kobe, Japan. University, Kyoto,
Japan.
Reactive oxygen species (ROS) including superoxide anions (O2-) play a key role in atherogenesis, and endothelial cells have the ability to generate ROS. To investigate the enzymatic sources of ROS and the effects of lysophosphatidylcholine (LPC), an atherogenic lipid, we measured ROS production in cultured bovine aortic endothelial cells (BAECs) by the lucigenin-enhanced chemiluminescence (CL) method and electron spin resonance (ESR). BAEC homogenates had the enzymatic activity of NADH/NADPH oxidase. BAECs cultured on microcarrier beads generated O2under basal conditions. The inhibition of NADH/ NADPH oxidase by diphenylene iodonium (DPI) significantly attenuated O2production, whereas no inhibitors of other oxidases suppressed it. Although LPC enhanced O2 production approximately 3.1-fold, its action was suppressed by DPI. Tyrosine kinase inhibitors significantly attenuated LPC-induced O2- production. ESR with DMPO demonstrated that LPC increased the formation of the DMPO-hydroxyl adduct in dose- and time-dependent manners. These data suggest that the basal production of O2- in endothelial cells is mainly mediated by the NADH/NADPH oxidase system and that LPC activates this oxidase to enhance O2- production through a tyrosine kinase-dependent pathway. The enhancement of ROS production by LPC is probably involved in its atherogenic property. J Atheroscler Thromb, 2000 ; 7 : 238-246. Key words : Oxidized LDL, Oxidant stress, Reactive oxygen species,
Introduction Oxidative stress by reactive oxygen species (ROS) in the vessel wall plays a key role in the development of atherosclerosis. Among the ROS, superoxide anions (O2-) are involved in atherogenesis by several mechanisms, including direct injury of the endothelium, oxidation of low density lipoprotein (LDL), inactivation of endothelium-derived relaxing factor (EDRF), and induction of the redox-sensitive gene such as vascular cell adhesion Address for correspondence : Nobutaka Inoue, MD, PhD, First Department of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail :
[email protected] Received April 3, 2000. Accepted for publication December 13, 2000.
Endothelium
molecule (VCAM-1) and monocyte chemoattractant protein (MCP-1) (1,2). Furthermore, O2- rapidly inactivates NO, and O2- and NO react with each other to generate peroxynitrite anion (ONOO-), which is considered to be one of the pathogens of atherosclerosis (3). The generation of ROS increases under various pathological conditions such as hyperlipidemia (4), hypertension (5), diabetes mellitus (6), and heart failure (7), and may consequently contribute to endothelial dysfunction. In the vessel wall, ROS are produced at several locations, including vascular smooth muscle cells (8, 9), endothelial cells (10, 11), and adventitial fibroblasts (12). The intracellular origins of ROS in endothelial cells and the regulatory mechanism of ROS production under pathological conditions are, however, poorly understood. Oxidized low-density lipoprotein (LDL) has been proposed to be a crucial factor in atherogenesis. Lyso-
LPC
Enhances
Superoxide
Production
phosphatidylcholine (LPC), which accumulates in oxidized LDL (13), increases in atherosclerotic lesions (14). Accumulating evidences indicate that LPC imparts various atherogenic properties to endothelial cells. For example, LPC inhibits endothelium-dependent vasorelaxation (15, 16), acts as a selective chemoattractant to mononuclear leukocytes (17), and induces expression of adhesion molecules such as intercellular adhesion molecules (ICAM-1), VCAM-1, P-selectin (18, 19). LPC also activates nuclear factor-kB (NF-kB), a redox-sensitive transcriptional factor, in endothelial cells (20, 21). Furthermore, LPC increases vascular O2- production in segments of the rabbit aorta (22). Given the importance of oxidative stress, the investigation of the regulatory mechanisms of endothelial production of ROS by atherogenic lipids such as LPC might provide important insights into understanding the pathogenesis of atherosclerosis. In the present study, we investigated the production capability and intracellular origins of ROS in cultured endothelial cells by lucigenin-enhanced chemiluminescence (CL) and electron spin resonance (ESR), and examined the effects of LPC on ROS production and its underlying signal transduction.
in Endothelial
The
activity
of O2-
lucigenin-enhanced reaction
50
(ALOKA,
mM
acid
pH
either
Cell
donor. was
scraping
excised
endothelial the
from
internal
a
described
(23,
modified
Eagle's
on
2.5 •~
106
and
Cytodex
microcarrier ous
stirring
in
The
final
volume
emission
was
signal
was
(C.P.M.)
20
supplemented
6 to
with
cultured
2.5 •~
on
107 cells
(24,
flask
became
used
The
4 days.
for
the
BAECs
of O2-
by
BAECs
were
1 mM
sion
was ice.
of O2beads
BAECs
4•Ž.
were
and ml
the of
lysis
Tris/HCI
an
The
cell
were
determined
serum
albumin
bovine
experiments,
centrifugation.
(membrane
(cytosol
and
g for
is
fraction)
was
was
resuspended
the
KCI,
15 min
BAECs
before
cultured
on chemi-
and
5.5
37°C
the
presence
in
and
of
phospholipids,
stirring
in
the
these
(PC)
a spinner
for
was 250
per
a
plateau.
CL
signal
agents. were
After
signal
count
reaching
BAECs
phosphatidylcholine
CL
containing
A23187,
1 mM
pH 7.4.
the
average
after and
10
CaCl2,
glucose,
the
microcar-
containing
HEPES-PSS
periods tiron
on
HEPES-buffered
1 mM
10 min,
as
using
with
NaCI,
mM
for in
10 min
cultured
times
mM
expressed
using
of the
In was
In
experi-
pretreated
2 hours
At IM
minute
with
with
contin-
flask.
by
as
at
removed, in 1
a
FR-20
Japan)
spin
at
room
width,
min
; and
output
used
as
a standard,
expressed
as
agent
was
ESR
the
100
g for
5
HEPES-PSS.
5.4 •~
mT
transferred
monitor
10
recorded
(X-band)
(JEOL,
under 100
; time
and
the kHz
relative
constant, 4 mW. the
Mn ESR
intensity.
into
was
; scanning
power
the
culture
stopping at in
spectrum
frequency,
500
After
centrifuged suspended
temperature
0.1
gain,
trap
radical
: modulation
receiver
and
gently
from
6 cells)
5, 5-dimethyl-1-pyroline-N-oxide
The
free
trypsin. were
were
harvested
(approximately
mM
cell.
were
with cells
suspension
flat
monolayers
cells
twice,
90
trapping
BAEC
and
the
washed cell
JES
ESR-spin
incubation
reaction,
(DMPO)
cytosol
by study,
PBS,
containing
tude
min
ROS ESR
with
dishes
tions
cell
60
for
(HEPES-PSS)
135
recorded
measured
stand-
Crude
100,000
7.4),
at
for
quartz
sec) by
at
fraction)
2
suspen-
as
membrane
centrifuged
(pH
(4 •~15
in
three
solution
(pH
and
The
lysis
fluoride,
ultrasonicator
by
buffer.
mM
phenylmethylsulfonyl
with
supernatant pellet
(50
with
of
homogenates
lucigenin-enhanced
2.8 •~107)
washed
5 mM
minutes,
phos-
scraped
by
salt
HEPES
trypsin
with
were
The
gently
A).
fractionated
homogenates
and
2 ,ƒÊM pepstatin
In some
of
ice-cold
reported
absence
cell
production
(approximately
beads
For
homogenates
with
CL
minute
luminescence
Detection
Cells
experiments.
concentrations
Bradford
protein.
times
inhibitors
500 ƒÊM
and
Protein of
were
(PBS),
homogenized
method ard
three
protease
EDTA,
leupeptin,
μM on
saline
containing
7.4),
in
per
the
agents
The
a
chemiluminescence
washed
phate-buffered buffer
production
lucigenin-enhanced
various
Photon
min.
were
in
reac-
1 ml.
20
data
experiments,
with
microcarrier
washed Measurement
some
the
count
CL
electron
homogenates.
was
observed
and
the
10 min, the
for
All
CL
83mM
acceptor
for
,u1 of
average
periods.
Measurement
uous
with
the
MgCl2,
as
solution
as
preincubated
LPC
continu-
coated
within
13 were
25). by
contained
measurement.
ments
seeded
37•Ž
100
recorded
the
experiments
or
lucigenin
In
(C.P.M.) 15%
dishes
were
beads
monolayer
passages
previously
of
mM
electron NADPH
at
adding
min
homogenates.
lucigenin
Dulbecco's
in suspension
spinner
cellular
between
then
as in
light
in a CL
solution
6.5
the
100 ƒÊM
by
subtracting
continuously
aorta
or
expressed
for
after
EDTA,
continuously
preincubation
isolated
thoracic
grown
serum,
maintained a
the
3 microcarrier
beads
confluent
calf
were
cow
were (DMEM)
fetal beads,
of
slaughtered
BAECs medium
heat-inactivated
(BAECs)
surface
freshly
24).
microcarrier
cells
by
The
detected
assay
as
preincubation
started
MgCl2, aortic
was
The
1 mM
NADH
After
tion
mM
culture
Bovine by
Methods
measured (CL).
lucigenin
lucigenin
100 ƒÊM
physiological and
7.4),
250 ƒÊM
were
was
N-2-hydroxyethylpiperazine-N7-2-ethanesulfonic
sucrose,
rier Materials
and
BLR-201).
(HEPES,
CL
production chemiluminescence
between O2-
reader
239
Cells
ESR with
following
condiampli-
367.5+7.5
0.1 sec
; sweep
(II) doped from
a
Tokyo,
; modulation
field,
signal
an
mT time,
in MgO
was
BAECs
was
; 2
240
Takeshita
Materials
A
Unless
otherwise
chased
from
chased
specified,
Sigma
from
Chemical.
Pharmacia
phenyliodonium and
GIBCO
Co.
Cytodex
DMPO
3
pur-
was
purdi-
Chemical.
A were
was
were
Biotechnology, Ro31-8220,
obtained
Corporation,
BRL.
Statistical
reagents
Aldrich
herbimycin
chem-Novabiochem
all
LKB
from
GF109203X
and
a
product
are
mean
from
genistein of
Calbiowas
Labotech,
from Japan.
analysis
Values
in
analysis
the
was
values
et al.
figures
carried
< 0.05
were
out
using
+SE, an
considered
and
statistical
unpaired
t-test.
P
significant.
B Results Effect
of
NADH
and
chemiluminescence Since to
be
the an
cells
important
of
and
of
1B,
preincubation
(n = 3,
P
