From the
Endothelial synthesis of NO is catalyzed by constitutive NO
synthase type III (NOS-III). NOS-III has been thought to be regulated
mainly at the level of enzyme activity by intracellular calcium. We
report that in human umbilical vein endothelial cells
lysophosphatidylcholine (lyso-PC), a component of atherogenic
lipoproteins and atherosclerotic lesions, increases NOS-III mRNA and
protein levels. This leads to the augmentation of NOS-III activity and
the enhancement of anti-platelet properties of endothelial cells.
Importantly, nuclear run-off experiments demonstrate a transcriptional
mechanism of regulation of NOS-III expression by
lysophosphatidylcholine. As endothelium-derived NO appears to be an
anti-atherogenic molecule, induction of NOS-III by lyso-PC may be a
protective response that limits the progress of the atherosclerotic
lesion and promotes its regression.
Endothelium-derived NO is a key molecule regulating various
physiological processes occurring at the interphase between the blood
and vascular wall(1) . Endothelial synthesis of NO is catalyzed
by constitutively expressed calcium- and calmodulin-dependent NO
synthase type III (NOS-III)
Although under certain conditions high
levels of NO may induce tissue injury(11) , the inhibitory
actions of endothelium-derived NO on vascular tone(12) ,
platelet activation(13) , leukocyte adhesion(14) ,
vascular smooth muscle cell proliferation, and de-differentiation (15, 16) suggest that NO is a protective and
anti-atherogenic molecule(17) . Likewise, inhibitors of NOS
enhance atherosclerosis in hypercholesterolemic
rabbits(18, 19) , whereas L-arginine, a
substrate for NOS, has a protective effect(20) .
Several
lines of evidence suggest a role for lysophosphatidylcholine (lyso-PC)
in atherogenesis(21) . Lyso-PC content of atherosclerotic
arteries is severalfold higher than that of normal vessels(22) .
Following pro-atherogenic modification of low density lipoproteins
lyso-PC may constitute up to 40% of their total lipid
content(23) . Lyso-PC is a chemoattractant for human
monocytes(24, 25) . Lyso-PC induces monocytic cell
expression of heparin binding epidermal growth factor(26) .
Importantly, lyso-PC causes induction of several endothelial genes
expressed in early atherosclerosis, such as vascular adhesion
molecule-1, intercellular adhesion molecule-1, platelet-derived growth
factor chains A and B, and heparin-binding epidermal growth
factor(27, 28) .
In this communication we present
experimental data that suggest that lyso-PC may also induce
vasoprotective endothelial genes. Our findings demonstrate that lyso-PC
enhances NOS-III gene transcription in cultured human umbilical vein
endothelial cells.
Nuclear Run-Off-Experiments were performed according
to an established laboratory procedure(31) . Nuclei
(10
Lyso-PC (30-100 µM) caused a time- and
concentrationdependent increase of NOS-III mRNA levels in cultured
human umbilical vein endothelial cells (HUVEC) (Fig. 1, A and B). NOS-III mRNA levels reached a maximum (11
± 2-fold increase by densitometry, n = 7) 6 h
after the stimulation with lyso-PC (100 µM).
Phosphatidylcholine (100 µM) was without effect (n = 2, not shown). The long half-life of NOS-III mRNA in
HUVEC (6) suggested that lyso-PC may enhance transcription of
the NOS-III gene rather then stabilize its mRNA. Indeed, actinomycin D
(5 µM) abolished lyso-PC-induced increases of NOS-III mRNA
levels (n = 2, not shown). Consistently, in three
separate nuclear run-off experiments, treatment of HUVEC with lyso-PC
(100 µM) for 3 h enhanced the rate of NOS-III mRNA
synthesis by isolated nuclei (Fig. 2, A and B).
This provides direct evidence for transcriptional activation of the
NOS-III gene by lyso-PC. The induction by lyso-PC of NOS-III mRNA in
HUVEC was not inhibited by cycloheximide, an inhibitor of protein
synthesis (Fig. 2C). We investigated the possibility
that the elevation of intracellular concentration of Ca
The major finding of this paper is the identification of
lyso-PC as a transcriptional inducer of constitutive endothelial
NOS-III. Our results demonstrate that in cultured HUVEC, lyso-PC causes
significant (11-fold) time- and concentration-dependent increase of
NOS-III mRNA levels. Increases of NOS-III mRNA levels are accompanied
by corresponding elevations (5.5-fold) of NOS-III protein levels. This
results in a 2-fold increase of NOS-III activity in HUVEC lysates. At
present, we do not know the basis of discrepancies between the levels
and time courses of lyso-PC-induced changes of NOS-III mRNA and protein
levels and those of NOS activity. These discrepancies suggest, however,
that the regulation of NOS-III expression in HUVEC is complex and that
it may also involve a lyso-PC-induced post-translational modification (e.g. phosphorylation) of NOS-III. Tetrahydrobiopterin has
recently been demonstrated to have a structural role in NO synthases
(37). Therefore, it is also possible that low levels of
tetrahydrobiopterin present in cultured HUVEC (38, 39) result in the inability of HUVEC to form active
NOS-III in spite of increased synthesis of NOS-III peptide.
Our
nuclear run-off experiments and the inhibitory effect of actinomycin D
demonstrate that the induction of NOS-III expression by lyso-PC is at
least in part mediated by the transcriptional activation of the NOS-III
gene. The major implication of these results is that the commonly held
view of NOS-III as a ``housekeeping gene'' regulated mainly
at the level of enzyme activity should be revised. Increased de
novo synthesis of NOS-III via induction of gene expression may be
an important mechanism that augments vasoprotective properties of
endothelium in response to extracellular insults. To the best of our
knowledge, lyso-PC is the first molecule to be shown to enhance the
transcription of the NOS-III gene. Whether signals other then lyso-PC
are also capable of transcriptional induction of NOS-III remains to be
elucidated. However, recent studies showing the enhancement of NOS-III
mRNA levels in cultured cells by shear stress (7) and in vivo by chronic exercise or estrogens(8, 9) suggest
that the regulation of NOS-III at the transcriptional level may be a
more common phenomenon. Like the induction by lyso-PC of
heparin-binding epidermal growth factor mRNA in human
monocytes(26) , induction of NOS-III mRNA levels was independent
of new protein synthesis. This indicates that the activation of the
NOS-III gene by lyso-PC involves the activation of a pre-existing
transcriptional factor or factors. We investigated the possibility that
the elevation of intracellular concentration of Ca
We have also conducted
experiments designed to determine whether the treatment of HUVEC with
lyso-PC potentiates the ability of HUVEC to inhibit platelet
aggregation, a key physiological function of endothelial cells. As
predicted, the treatment of HUVEC with lyso-PC reduced the number of
HUVEC required to inhibit aggregation of human washed platelets. The
effect of lyso-PC was reversed by an inhibitor of NO synthase. These
results demonstrate that lyso-PC augments the anti-platelet properties
of HUVEC and, considered together with the evidence for the
simultaneous induction of NOS-III expression, suggest the induction of
NOS-III as a mechanism involved. However, whether this is the only
mechanism remains to be established.
Identification of lyso-PC as an
inducer of the NOS-III gene raises several intriguing questions
regarding the biological significance of lyso-PC-triggered induction of
NOS-III. It is well established that both in experimental models of
atherosclerosis (44, 45, 46, 47) and in
human disease (48, 49, 50, 51) atherosclerosis leads
to the impairment of endothelium-dependent relaxation, which in most
vascular beds is mediated by endothelium-derived NO. It has also been
convincingly demonstrated by several laboratories that in vitro incubation of isolated blood vessels with oxidized LDL leads to
the impairment of endothelium-dependent relaxation similar to that
observed in atherosclerotic
vessels(52, 53, 54, 55) . Lyso-PC has
been identified as the critical diffusible component of oxidized LDL
that is responsible for a rapid inhibition of endothelium-dependent
relaxation by oxidized LDL(54, 55) . It has also been
recently reported that chemically oxidized LDLs reduce NOS-III mRNA
levels in human endothelial cell via destabilization of NOS-III
mRNA(56) . Thus, in the context of the studies summarized above,
augmentation of endothelial expression and activity of NOS-III by
lyso-PC comes as a surprise and may at first appear to be
contradictory. However, a careful analysis demonstrates that these
results are not necessary conflicting. It should be emphasized that the
reduction of endothelium-dependent relaxation in isolated vessel can be
due to factors other then the reduction of endothelial capacity to
synthesize NO. For instance, the impairment of endothelium-dependent
relaxation could be caused by the reduction of the half-life of NO in
atherosclerotic vessels. Indeed, oxidized LDLs have been demonstrated
to reduce the half-life of NO in
vitro(57, 58) , and both hypercholesterolemia in
experimental animals (59) and lyso-PC in isolated blood vessels (60) reduce the half-life of NO via induction of superoxide
anion generation. Importantly, intimal thickening, which is a typical
characteristic of atherosclerotic vessels, can be expected to decrease
the amount of endothelium-derived NO that reaches vascular smooth
muscle. Thus, it is possible that the impairment of
endothelium-dependent relaxation and the augmentation of endothelial
synthesis of NO co-exist. In this context, it is of interest that the
release of NO measured by a chemiluminescent technique is increased in
aortas from hypercholesterolemic rabbits (61). Recently, enhancement of
the release of NO from cultured rabbit endothelial cells by oxidized
LDL has been reported(62) . Moreover, intensive staining for
NOS-III has been observed in atherosclerotic vessels(5) .
Interestingly, the same paper that demonstrated that chemically
oxidized LDLs decrease NOS-III mRNA levels via reduction of NOS mRNA
half-life (56) also showed data demonstrating that oxidized LDLs
double the NOS-III transcription rate. In the same paper, although not
discussed in detail by the authors, results were presented that showed
that the incubation of native LDL with endothelial cells, which results
in oxidative modification of LDL, evidenced by increased content of
thiobarbituric acid reactive substances, results in the enhancement of
NOS-III mRNA levels. Thus, it is tempting to speculate that the
increase of NOS-III mRNA levels by endothelial cell-oxidized LDL and
the augmentation of the NOS-III transcription rate by chemically
oxidized LDL may be due to lyso-PC-induced activation of the NOS-III
gene. It is possible that chemically modified LDL has additional
effects on endothelial cells that cause destabilization of NOS-III
mRNA.
As outlined in detail in the Introduction, lyso-PC causes
induction of several potentially pro-atherogenic endothelial genes
involved in leukocyte recruitment, mitogenesis, and
inflammation(27, 28) . Our findings add another
inducible protein to this growing list. However, this may be an
important addition, as in contrast to other genes showed to be induced
by lyso-PC so far, the biological actions of endothelium derived NO
suggest that NOS-III is a key vasoprotective gene. These findings are
intriguing because they introduce a novel idea that a single molecule
can induce both pro-atherogenic and vasoprotective mechanisms. The
attractiveness of this hypothesis stems from the fact that it may help
to rationalize several poorly understood issues concerning
atherogenesis. Induction of both pro-atherogenic and vasoprotective
mechanisms may explain a long time course of atherosclerotic lesions
development. This may also explain the potential of early
atherosclerotic lesions to regress after removing of initiating
environmental factors. We acknowledge, however, that further work,
beyond the limitations of the present study, is required to verify the
significance of lyso-PC-induced activation of the NOS-III gene in the
proper pathophysiological setting.
In conclusion, our results
demonstrate that lyso-PC enhances expression of NOS-III in HUVEC by a
transcriptional mechanism. Induction of NOS-III by lyso-PC may be a
protective response that limits the progress of the atherosclerotic
lesion and promotes its regression.
We thank D. Loose-Mitchell for review of the
manuscript, P.-F. Chen for cloning NOS-III, Sang Lee and J. Juneja for
supplying HUVEC, W. C. Sessa for NOS-III transfected cell line, T.
Scott-Burden for human aortic vascular smooth muscle cells, and M.
Kruzel for
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(2) . In
contrast to calcium-independent NOS type II that can be induced in many
cell types by cytokines, NOS-III has been thought to be regulated
mainly by changes in the intracellular concentrations of calcium
(3-5). The importance of regulation of NOS-III expression as a
means of regulation of endothelial synthesis of NO production has only
recently emerged as a possibility. It has been shown that NOS-III mRNA
levels in cultured human umbilical vein endothelial cells are reduced
by tumor necrosis factor-
and that this is due to de-stabilization
of NOS-III mRNA(6) . Several recent reports also indicated that
NOS-III expression could be up-regulated. Increased levels of NOS-III
mRNA levels have been reported in bovine endothelial cells subjected to
shear stress(7) , in aortas isolated form dogs undergoing
exercise training(8) , and in guinea pigs following treatment
with estrogens (9). However, in no case mechanisms leading to the
increase of NOS-III mRNA levels have been elucidated. Recent cloning of
the human NOS-III gene revealed the presence in a putative promoter
region of the NOS-III gene of several potentially cis-acting regulatory
elements which in other genes have been demonstrated to regulate gene
expression in response to cAMP, cholesterol, protein kinase C
activation, transforming growth factor
, and shear
stress(10) . Whether any of these regulatory elements is
involved in regulation of the NOS-III gene in response to extracellular
stimulation has not been established yet. In fact, no molecule has been
conclusively demonstrated to induce NOS-III expression at the level of
gene transcription so far.
Materials
Lyso-PC
(1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) was from
Avanti Polar Lipids (Birmingham, AL). Unless otherwise indicated all
the other reagents were from Sigma.
Tissue Culture
HUVEC were cultured as described
previously (29) in Medium 199 containing 20% bovine calf serum
(Hyclone, Logon, UT), 12.5 µg/ml endothelium cell mitogen
(Biomedical Technologies, Stoughton, MA), and 100 µg/ml heparin.
Only second and third passage cells were used. Twelve hours before
lyso-PC treatment cells were transferred to Medium 199 containing 5%
fetal bovine serum and no other additives.
Northern Blotting
Total RNA was isolated using
Ultraspec (Biotecx Laboratories, Houston, TX). 15-25 µg of
RNA was fractionated on 1% agarose and transferred to a positively
charged nylon membrane. As NOS-III probe we used a 1.6-kilobase BglII/EcoRI restriction fragment of full-length human
NOS-III cDNA cloned in our laboratory (30) and subcloned into EcoRI sites of pcDNA-3 (Invitrogen). Probe labeling,
hybridization, and chemiluminescent detection were performed using
components of Genius system (Boehringer Mannheim). Hybridization
conditions (5 SSC, 0.02% SDS, 0.1% sarcosyl, 2% blocking
reagent, 68 °C, overnight) and high stringency washes (0.1
SSC, 0.1% SDS, 60 °C, 45 min) assured no cross-hybridization with
any other HUVEC mRNA or NOS-II mRNA induced in cultured human aortic
vascular smooth muscle cells by 12-h stimulation with human
interleukin-1
(100 pg/ml) and
-interferon (250 units/ml) (n = 2). Membranes were stripped by boiling in 0.1
SSC containing 1% SDS for 15 min and re-hybridized to
5`-digoxigenin-labeled nucleotide, 5`-ACGGTATCTGATCGTCTTCGAACC-3`,
complementary to human 18 S ribosomal RNA or/and to a
digoxigenin-labeled glyceraldehyde-3-phosphate dehydrogenase cDNA
probe. Blots were quantified using the Bio Image system (MilliGene).
), isolated from three to five T75 flasks of confluent
HUVEC, were incubated in the presence of 0.25 mCi of
[
P]GTP and other unlabeled nucleotides (1
mM) in a volume of 200 µl at 26 °C for 25 min.
Transcribed RNAs were isolated using Ultraspec and equal amounts
(6-8
10
cpm) were hybridized to denatured
plasmids (15 µg) containing cDNAs of NOS-III and chloramphenicol
acetyltransferase (CAT) (both in pcDNA-3) or hamster
tubulin cDNA
in pAcUW51 (Pharmingen) immobilized on nitrocellulose membranes.
Conditions for hybridization and washes were identical to those during
Northern blotting. Densities of bands were quantified using the Bio
Image system (MilliGene). To normalize the results, the intensities of
-tubulin bands were assigned an arbitrary densitometric unit of
one. Intensities of other bands were expressed as a fraction of the
density of
-tubulin bands.
Western Blotting
Following incubation with lyso-PC
for the desired times, HUVEC were washed with ice-cold
phosphate-buffered saline containing 0.5 mM EDTA and lysed in
300 µl of buffer containing 0.05 M Tris-HCl (pH 7.4), 1%
Nonidet P-40, 60 mML-arginine HCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 100
µM tetrahydrobiopterin, and all components of Boehringer
Mannheim Protease Inhibitors Set. Following centrifugation at 13,000
g for 2 min, the protein concentrations in lysates
were determined using Bio-Rad DC protein assay. Lysates (15 µg of
protein) were analyzed by electrophoresis on 7% SDS-polyacrylamide gels
and electroblotted onto nitrocellulose membranes. Membranes were probed
using monoclonal antibodies from Transduction Laboratories (Lexington,
Kentucky). Antibody N30020 is selective for NOS-III, and antibody
N32020 binds to all isoforms of NOS. In some experiments, to confirm
the specificities of antibodies, NOS-III standard (1 µg of lysate
of stably transfected human kidney cell line 293 overexpressing bovine
NOS-III) and NOS-II standard (4 µg of lysate of human aortic
vascular smooth muscle cells stimulated with 100 pg/ml human
interleukin-1
and 250 units/ml
-interferon for 24 h) were
also analyzed. Immunoreactive bands were visualized using ECL system
(Amersham Corp.).
NOS Activity Assay
NOS activity was assayed in the
same lysates that were used for Western blotting. NOS activity was
determined as L-NAME-inhibitable conversion of L-arginine to L-citrulline, as described
previously(32) . Incubations were carried out with or without L-NAME (300 µM) and in the presence of
150-200 µg of protein, 10 cpm
[2,3-
H]L-arginine HCl, 60 µM unlabeled L-arginine HCl, 10 mM NADPH, 1
mM dithiothreitol, 100 nM calmodulin, 1 mM Ca
, and 100 µM tetrahydrobiopterin
for 30 min.
Platelet Aggregation
Human washed platelets were
prepared as described previously(33) . Anti-platelet properties
of HUVEC were assayed using an adaptation of an established method of
bioassay of NO (34). HUVEC were harvested using trypsin/EDTA into
ice-cold Tyrode buffer composed of 8 g/liter NaCl, 0.2 g/liter KCl,
0.225 g/liter MgCl
6H
O, 0.05 g/liter
NaH
PO
, 1 g/liter NaHCO
, 1.5 g/liter
glucose. Typically, equal numbers of control and lyso-PC-treated cells
were isolated and 70-80% of those excluded trypan blue. Platelet
aggregation was studied using a light transmission aggregometer
(Chronolog) in the volume of 500 µl. Incubations contained 2.5
10
platelets/ml suspended in Tyrode buffer
containing 1 mM Ca
. HUVEC suspensions or the
corresponding volumes of Tyrode buffer (3-20 µl) were added
to stirred platelets 2 min before thrombin. L-NAME was added 1
min before the addition of HUVEC.
or activation of protein kinase C, known actions of lyso-PC in
endothelial cells(35, 36) , mediates the induction of
NOS-III. However, 2-12-h stimulation of HUVEC with calcium
ionophore A23187 (3 µM), phorbol myristate acetate (0.3
µM), or their combination did not increase NOS-III mRNA
levels (n = 3, not shown).
Figure 1:
Induction of
NOS-III mRNA expression in HUVEC by lyso-PC. A, Northern
blotting analysis of 25 µg of total RNA isolated from control HUVEC (lanes 1-3) and HUVEC stimulated with lyso-PC (100
µM: lanes 4, 6, and 7) for 3, 6, and 12
h using digoxigenin-labeled NOS-III cDNA probe. Lane 5 was not
loaded. This blot was re-hybridized using ribosomal 18 S RNA probe. B, Northern blotting analysis of 20 µg of total RNA
isolated from HUVEC incubated in the absence (lane 1) and the
presence of 30 µM, 60 µM, and 100 µM (lanes 2-4) concentrations of lyso-PC. This blot
was re-hybridized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.
Figure 2:
Induction of NOS-III gene transcription in
HUVEC by lyso-PC. A, nuclear run-off analysis of transcription
rates of NOS-III and -tubulin (
-Tub) genes in
control HUVEC and HUVEC stimulated with lyso-PC (100 µM)
for 3 h. Background hybridization to the CAT-pcDNA-3 plasmid is also
shown. B, Densitometric analysis of the autoradiograph shown
in A. Intensities of
-tubulin (
-Tub) bands
were assigned an arbitrary densitometric unit of 1. This graph shows a 2.0-fold increase of NOS-III band intensity in the blot
hybridized to RNA synthesized by nuclei isolated from lyso-PC-treated
HUVEC. In two other experiments performed similar analysis revealed
2.0- and 2.15-fold increases. Intensities of NOS-III and pcDNA-3 bands
in control were identical. This indicates that the basal transcription
of the NOS-III gene was below the level of detection of the assay. This
is not unexpected, considering low levels of NOS-III mRNA and its
stability in HUVEC. Therefore, densitometric analysis may underestimate
the actual level of induction of NOS-III gene transcription by lyso-PC. C, Northern blot analysis of 20 µg of total RNA isolated
from control HUVEC (lane 1) and HUVEC incubated for 6 h with
cycloheximide (CHEX, 10 µM; lane 2),
lyso-PC (100 µM; lane 3), or their combination (LysoPC&CHEX; lane 4).
In agreement with changes
of NOS-III mRNA levels, lyso-PC (100 µM) caused a
time-dependent increase of NOS-III protein in HUVEC lysates (Fig. 3A). NOS-III levels reached a peak (5.5 ±
1.5 -fold increase by densitometry, n = 3) 12 h
following stimulation with lyso-PC (100 µM). In the same
lysates, lyso-PC induced a biphasic change in NOS activity (Fig. 3B). NOS activity decreased to 42 ± 6% of
control (n = 4, p < 0.05) 3 h after
stimulation with lyso-PC. At later time points NOS activity increased
and reached a plateau after 12 h at the level 2-fold above that in
control lysates (Fig. 3B). We found no evidence for
NOS-II expression in HUVEC by immunoblotting (Fig. 3A).
Our NOS-III antibody recognized only the NOS-III protein expressed in
HUVEC and the NOS-III protein standard. The slight difference in
electrophoretic mobility of NOS-III of HUVEC and that of NOS-III
standard is due to species differences. The NOS-III standard contained
bovine NOS-III. Consistently, NOS activity was abolished in the absence
of Ca (100% inhibition, n = 2), a
property of NOS-III but not NOS-II.
Figure 3:
Induction of NOS-III protein and NOS-III
activity in HUVEC by lyso-PC. A, Western blotting analysis of
NOS-III in HUVEC lysates 0, 3, 6, 12, and 24 h (lanes
1-5) following stimulation with lyso-PC (100
µM). NOS-III standard (1 µg of lysate of stably
transfected human kidney cell line 293 overexpressing bovine NOS-III, lane 6) and NOS-II standard (4 µg of lysate of human
aortic vascular smooth muscle cells stimulated with 100 pg/ml human
interleukin-1 and 250 units/ml
-interferon for 24 h, lane
7) were also analyzed. NOS monoclonal antibodies were from
Transduction Laboratories. NOS-III-specific antibody (N30020)
recognized a single 135-kDa band in HUVEC lysates and the NOS-III
standard, but not in the NOS-II standard. Below is shown the same
membrane stripped and reprobed with antibody (N32020) that binds to all
three isoforms of NOS. It revealed the presence of the 130-kDa NOS II
band in the NOS-II standard. This blot was intentionally overexposed.
In spite of that, no NOS-II could be detected in HUVEC. B,
time course of NOS-III protein expression and activity (18) in HUVEC
lysates. NOS activity in control HUVEC was 2.1 ± 0.4 pmol/mg of
protein/min (n = 4). Points represent means ±
S.E. of three to four experiments.
To determine whether induction
of NOS-III by lyso-PC has the expected physiological consequences, we
evaluated the effect of treatment with lyso-PC on the anti-platelet
properties of HUVEC. We used a modification of the established method
developed to bioassay the release of NO from endothelial cells. As
predicted, treatment of HUVEC with lyso-PC (100 µM) for 24
h decreased the number of HUVEC required to inhibit thrombin-induced
aggregation of human washed platelets (Fig. 4). The effect of
lyso-PC was reversed by NOS inhibitor, L-NAME.
Figure 4:
Augmentation of anti-platelet properties
of HUVEC by lyso-PC. The figure shows superimposed platelet aggregation
traces from an experiment in which the effect of control and
lyso-PC-treated HUVEC on aggregation of human washed platelets induced
by thrombin (Thr, 40 milliunits/ml) was investigated. In this
experiment, 10 control HUVEC was required to inhibit
platelet aggregation by 50%. 0.25
10
cells were
without effect. Following incubation with lyso-PC (100 µM)
for 24 h, only 0.25
10
HUVEC was required to
inhibit platelet aggregation by 50%. The effect of 10
cells
is also shown. HUVEC-induced inhibition of platelet aggregation was
prevented by NOS inhibitor, L-NAME (300 µM). This
experiment was repeated three times with similar results. Number of
control HUVEC required to inhibit platelet aggregation by 50% varied in
each experiment from 10
to
10
.
or
activation of protein kinase C, known actions of lyso-PC in endothelial
cells(35, 36) , mediates the induction of NOS-III.
However, calcium ionophore A23187, phorbol myristate acetate, or their
combination did not increase NOS-III mRNA levels. Lyso-PC has been
shown to modify G-proteins (40) and activate adenylate
cyclase(41) . However, we did not explore a possibility that any
of these effects is involved in transduction pathway of the NOS-III
gene activation by lyso-PC. Thus, as in the case of other
lyso-PC-activated genes, the signal transduction pathway and
transcriptional factors involved in activation of the NOS-III gene by
lyso-PC remain to be established. It is also important to note that
lyso-PC is the major product of action of cellular phospholipases
A
on phosphatidylcholine, the most abundant
phospholipid(42) . In contrast to arachidonic acid, the other
product of action of phospholipases A
which is a substrate
for enzymes involved in the synthesis of prostanoids and leukotrienes,
a role of lyso-PC in signal transduction has only recently been
suggested(43) . Therefore, a putative role of intracellularly
generated lyso-PC in the regulation of NOS-III or other
lyso-PC-inducible genes in response to extracellular signals might be
an interesting area of investigation.
-nitro-L-arginine; LDL(s), low
density lipoprotein(s).
-tubulin plasmid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.