(Received for publication, July 25, 1994; and in revised form, November 3, 1994)
From the
The atherogenic effects of low-density lipoprotein (LDL) may be
mediated, in part, by its effect(s) on endothelial-derived nitric oxide
(NO). To determine whether LDL can modulate NO production by changing
NO synthase expression, we treated human saphenous vein endothelial
cells with increasing concentrations of native or oxidized LDL
(0-100 µg/ml) for various durations (0-72 h). Oxidized,
but not native LDL caused a time-dependent decrease in steady-state NO
synthase mRNA levels. This coincided with a maximal 56% decrease in NOS
activity as determined by [H]arginine to
[
H]citrulline conversion. In the presence of
actinomycin D, treatment with oxidized LDL reduced the half-life of NO
synthase mRNA from 36 to 10 h. This decrease in NO synthase mRNA
correlated with the degree of LDL oxidation and was attenuated by
pretreatment with cycloheximide. Nuclear run-off studies showed a
biphasic transcriptional pattern of NO synthase gene with an initial
25% decrease during the first 6 h followed by a maximal 2.2-fold
increase over baseline during the subsequent 18 h. These results
indicate that oxidized LDL regulates endothelial NOS expression through
a combination of early transcriptional inhibition and
post-transcriptional mRNA destabilization.
Nitric oxide (NO) is produced in endothelial cells by an enzyme
known as NO synthase which converts L-arginine to L-citrulline and NO(1, 2) . Endothelial NO
synthase is a constitutively expressed enzyme with relatively low basal
activity(3, 4) . Its activity, however, is rapidly
stimulated by increases in intracellular calcium through specific
signal tranduction pathways(5, 6) . Considerable
evidence indicates that atherogenic oxidized low-density lipoprotein
(LDL) ()can cause endothelial-dependent vasomotor
abnormalities through inhibition of endothelial-derived
NO(7, 8, 9) . Alteration of endothelial NO by
oxidized LDL has been shown to occur at the level of membrane signal
transduction(10) , NO production(11) , and NO
inactivation(12) . These findings are important since nitric
oxide can potentially inhibit several components of the atherogenic
process such as vascular smooth muscle cell proliferation(13) ,
platelet aggregation(14) , monocyte adhesion(15) , and
even oxidative modification of LDL(16) .
We have previously
reported that native LDL can attenuate bradykinin-stimulated
endothelial NO production via inhibition of specific guanine
nucleotide-binding proteins (G) which couple the bradykinin
receptor to NO synthase activation(17) . This is in contrast to
oxidized LDL which can inhibit NO production in response to the
non-receptor-dependent calcium ionophore
A23187(7, 8) . These findings suggest that oxidized
LDL differs from native LDL in being able to inhibit NO production at
the level of NO synthase. Indeed, recent studies have shown that
oxidized, but not native LDL can decrease the activity of the
cytokine-inducible NO synthase in murine macrophages without affecting
its steady-state mRNA or protein expression(18) . Similar
findings have not been reported for the constitutive NO synthase
isoform in endothelial cells. Furthermore, the effects of oxidized LDL
on the expression of endothelial NO synthase is not known. Since the
regulation of endothelial NO production by oxidized LDL may mediate
some of its atherogenic effects, we sought to characterize the effects
of oxidized LDL on endothelial NO synthase expression at the level of
transcriptional and post-transcriptional regulation.
The degree of LDL oxidation was estimated by
measuring the amounts of thiobarbituric acid reactive substances
(TBARS) produced using a colorimetric assay for
malondialdehyde(21) . LDL samples (50 µg) or supernatant
from cell incubation (500 µl) were mixed with 1 ml of
trichloroacetic acid (20%) and 1 ml of thiobarbituric acid (1%), and
heated at 100 °C for 30 min. After cooling in a water bath (22
°C), the mixture was centrifuged at 12,000 g for
15 min and the absorbance was measured at 535 nm (Beckman DU-64
Spectophotometer). Serial dilutions of 1,1,3,3-tetramethoxypropane
which yields malondialdehyde was used to construct the standard curve.
The extent of LDL modification was expressed as nanomole of
malondialdehyde per mg of LDL protein.
The supernatant was removed, and the cells were scraped and
lysed by a probe sonicator (Model W185F, Ultrasonics, Inc., Plainview,
NY). Approximately 4 ml of this cellular extract and supernatant was
applied to a column containing 2 ml of Dowex 50WX-8 resin
(pre-equilibrated with NaOH) followed by elution of
[H]citrulline with 2 ml of water. A sample of the
eluant (1 ml) was counted for 2 min in a liquid scintillation counter
(Beckman LS 1800). Preliminary studies showed that these Dowex columns
extracted >95% [
H]arginine and <8%
[
H]citrulline. Nonspecific activity was
determined by [
H]citrulline production in the
absence of A23187 stimulation and represented approximately 5% of total
activity. The Me
SO (0.1%) used to solubilized A23187 did
not produce appreciable changes (<1%) in baseline NO synthase
activity.
In vitro transcription using the nuclear
pellet (100 µl) was carried out in a shaking water bath at 30
°C for 30 min in a buffer containing 10 mM Tris-HCl, pH
8.0, 5 mM MgCl, 300 mM KCl, 50 µM EDTA, 1 mM dithiothreitol, 0.5 units of RNasin, 0.5
mM CTP, ATP, and GTP, and 250 µCi of
[
-
P]UTP. The reaction was terminated by
incubating the assay with 40 units of DNase I (Ambion, Austin, TX) for
20 min at 30 °C. Proteins in the mixture were degraded by a
solution containing 0.4% SDS, 40 mM Tris-HCl, pH 7.4, 10
mM EDTA, and 400 µg/ml proteinase K (E. Merck, Darmstadt,
W. Germany), and extracted by phenol/chloroform. Ammonium acetate (2 M) and an equal volume of EtOH were added to the radiolabeled
RNA transcripts. The mixture was kept in dry ice for 20 min prior to
precipitation by centrifugation at 12,000
g for 10
min.
Equal amounts (1 µg) of purified, denatured full-length
endothelial NO synthase, human -actin, and linearized pGEM-3z cDNA
were vacuum-transferred onto nylon membranes using a slot blot
apparatus (Schleicher & Schuell). The membranes were baked and
prehybridized as described for Northern blots. The precipitated
radiolabeled transcripts (
5
10
cpm) were
resuspended in 2 ml of hybridization buffer containing 50% formamide, 5
SSC, 2.5
Denhardt's solution, 25 mM sodium phosphate buffer, pH 6.5, 0.1% SDS, and 250 µg/ml
salmon sperm DNA. Hybridization of radiolabeled transcripts to the
nylon membranes was carried out at 45 °C for 48 h. The membranes
were then washed with 1
SSC, 0.1% SDS for 1 h at 65 °C
prior to autoradiography for 72 h at -80 °C.
Native LDL had an initial TBARS value of 0.1 ± 0.1
nmol/mg which increased to 0.6 ± 0.3, 2.3 ± 0.8, and 4.5
± 1.2 nmol/mg after 24, 48, and 72 h of incubation with
endothelial cells in Medium 199 and lipoprotein-deficient serum,
respectively. LDL samples which have been oxidized by exposure to
CuSO exhibited much higher levels of oxidative modification
with TBARS values ranging from 7.3 ± 1.4 nmol/mg after a 6-h
exposure with 5 µM CuSO4 to 48.8 ± 4.3 nmol/mg
after a 24-h exposure with 10 µM CuSO4.
Figure 1:
Northern blots (20 µg total
RNA/lane) showing the time course of NO synthase mRNA expression in
response to treatment with (A) native LDL (100 µg/ml) and (B)
oxidized LDL (50 µg/ml, TBARS 24.4 nmol/mg). RNA loading was
determined by hybridization to human -actin. This is
representative of three separate
experiments.
Figure 2:
Northern blots (10 µg total RNA/lane)
showing dose-dependent effects of oxidized LDL (TBARS 24.4 nmol/mg) at
24 h on NO synthase mRNA levels from primary human aortic endothelial
cells. Equal RNA loading for each experiment was verified by
hybridization to -actin. Experiments were performed two
times.
Figure 3:
NO
synthase mRNA expression in response to increasing oxidative
modification of LDL (50 µg/ml) as assessed by TBARS. Equal RNA
loading for each experiment was verified by hybridization to
-actin. Experiments were performed two
times.
The half-life of NO synthase mRNA was determined in the presence of actinomycin D (5 µg/ml). Oxidized LDL (50 µg/ml) shortened the half-life of NO synthase mRNA from 36 ± 6 to 10 ± 3 h (Fig. 4, A and B). This reduction in NO synthase half-life suggests that oxidized LDL caused post-transcriptional decreases in NO synthase mRNA stability. Pretreatment with cycloheximide (10 µg/ml), however, prevented the decrease in NO synthase mRNA prior to 18 h (Fig. 5, A and B). These findings indicate that the effect(s) of oxidized LDL on NO synthase mRNA destabilization requires new protein synthesis.
Figure 4: A, Northern blots (20 µg total RNA/lane) showing the effects of actinomycin D (ACT, 5 µg/ml) on NO synthase (NOS) mRNA in the presence and absence (control) of oxidized (ox) LDL (50 µg/ml; TBARS 24.4 nmol/mg). Corresponding ethidium bromide-stained blots did not reveal any significant differences in ribosomal 28 S band intensities. B, densitometric analysis of Northern blots showing NO synthase mRNA levels (relative intensity) as a semi-log function of time in the presence of actinomycin D alone or in combination with oxidized LDL.
Figure 5: A, Northern blots (10 µg total RNA/lane) showing the effects of oxidized (ox) LDL (50 µg/ml; TBARS 24.4 nmol/mg) on NO synthase (NOS) mRNA expression in the presence and absence of cycloheximide (CHX; 10 µg/ml). Equal RNA loading was verified by equal ribosomal 28 S band intensities. B, densitometric analysis of Northern blots showing NO synthase mRNA levels (relative intensity) as a semi-log function of time.
Figure 6:
NO synthase activity as determined by
calcium ionophore A23187 (0.1 µM)-stimulated
[H]arginine to
[
H]citrulline conversion. A, oxidized
LDL (50 µg/ml; TBARS 24.4 nmol/mg) time course; B,
oxidized LDL (TBARS 24.4 nmol/mg) dose-response after 24 h. Each
experiment was performed three times in
duplicate.
Similar decreases in NO synthase activity
were also observed with native and oxidized LDL (TBARS 24.4 nmol/mg) in
a concentration-dependent manner with maximal effect occurring at a
concentration of 50 µg/ml (Fig. 6B). After 24 h of
treatment, native LDL decreased NO synthase activity to 1.07 ±
0.05 pmol/min/million cells; whereas, oxidized LDL had a greater effect
in decreasing NO synthase activity to 0.65 ± 0.06
pmol/min/million cells. The calculated IC values for
native and oxidized LDL were 14.4 ± 0.18 and 11.2 ± 0.12
nmol/mg, respectively. Furthermore, decrease in NO synthase activity
also correlated with the degree of LDL oxidation. After exposure to LDL
(50 µg/ml) for 24 h, the calculated TBARS IC
value was
21.4 ± 3.8 nmol/mg (data not shown).
Figure 7:
Blots from a representative nuclear
run-off experiment showing the effects of oxidized LDL (50 µg/ml;
TBARS 24.4 nmol/mg) on time-dependent transcriptional activity of
-actin and NO synthase (NOS) gene. Band intensities of NO synthase
compared to
-actin (relative index) at the indicated time point
for three separate experiments are shown. Nonspecific activity was
determined by hybridization to pGEM vector.
Preliminary studies using different amounts of
radiolabeled RNA transcripts demonstrate that under our experimental
conditions, hybridization was linear and nonsaturable. The density of
each NO synthase band was standardized to the density of its
corresponding -actin band at each time point (relative index). To
exclude the possibility that changes in NO synthase relative index is
due to changes in
-actin transcription caused by oxidized LDL,
another marker,
-tubulin, was included on each of the nuclear
run-off blots. Similar relative indices were obtained when NO synthase
transcription was standardized to
-tubulin transcription (data not
shown). The specificity of each band was determined by the lack of
hybridization to the nonspecific pGEM cDNA vector.
In this study, we have characterized the effects of LDL on endothelial NO synthase mRNA expression and correlated these effects with decreases in NO production as assessed by L-arginine to L-citrulline conversion. We found that exposure to noncytotoxic concentrations of oxidized, but not native LDL caused a progressive time-dependent decrease in steady-state NO synthase mRNA levels. Furthermore, additional studies using human aortic endothelial cells yielded similar results. The mechanism(s) responsible for this decline is dependent upon oxidative modification of LDL and involves a combination of early transcriptional inhibition and predominantly, post-transcriptional mRNA degradation. This latter effect of oxidized LDL on NO synthase mRNA apparently requires the participation of newly synthesized protein(s) as demonstrated by studies using the protein synthesis inhibitor, cycloheximide. It is not known whether these transcriptional and post-transcriptional processes are regulated separately by oxidized LDL, and are therefore, perhaps differentially sensitive to specific characteristics of the LDL particle such as the concentration or the degree of modification of its constituents.
Increases in NO synthase mRNA degradation have been observed with
exposure to certain proinflammatory cytokines such as tumor necrosis
factor- and interleukin-1
(28, 29) . Both
tumor necrosis factor-
and interleukin-1
are associated with
atherogenesis and their presence has been demonstrated in
atherosclerotic lesions(30) . Endothelial cells can produce
interleukin-1
which in turn, can regulate diverse vascular
functions in an autocrine and paracrine fashion(31) . Analysis
of our endothelial NO synthase cDNA reveals a sequence (AUUUA) in the
3`-untranslated region which is known to be involved in mRNA
destabilization(32) . It remains to be determined whether this
sequence is utilized as the putative site of regulation by oxidized
lipids and cytokines. Since atherosclerotic vessels contain oxidized
LDL (33) and respond abnormally with respect to altered
endothelial NO activity(34) , it is interesting to speculate
whether translational induction of these cytokines by oxidized LDL is
the mechanism by which oxidized LDL destabilizes NO synthase mRNA.
The regulation of NO synthase gene transcription by oxidized LDL is
complex and consists of at least two separate components. Early
transcriptional inhibition occurring at 6 h is most probably due to
direct effects of oxidized LDL since small decreases in transcriptional
activity can be observed within 30-120 min of exposure to
oxidized LDL. ()The 5`-flanking promoter region of the human
constitutive endothelial NO synthase has recently been characterized
and found to contain a DNA binding sequence (GCGGGGCG) for an
inhibitory transcription factor(35) . It is not known whether
oxidized LDL represses NO synthase transcription through activation of
this DNA binding protein or through other yet unidentified silencer
elements. Increases in NO synthase transcriptional activity occurring
after 12 h is probably the result of indirect effects of oxidized LDL
or secondary to some feedback counter-regulatory mechanism induced by
declining NO synthase activity and steady-state mRNA levels. This
delayed increase in NO synthase transcription suggests translational
induction of new protein(s) as the most likely mechanism for enhanced
transcription although direct effects of oxidized LDL components with
delayed access into endothelial subcellular compartments cannot be
excluded. Nevertheless, despite a greater than 2-fold increase in
transcription rate, steady-state NO synthase mRNA levels continue to
fall after 12 h as a result of an even higher rate of NO synthase mRNA
degradation. This marked effect on mRNA stability is evident by the
more than 3-fold reduction in NO synthase half-life as determined in
the presence of the transcriptional inhibitor, actinomycin D.
Despite increases in NO synthase mRNA after exposure to native LDL, NO synthase activity remains mildly reduced. This suggests that native LDL can also inhibit NO production by either decreasing NO synthase protein expression or attenuating its enzymatic activity. This is in agreement with previous studies showing that short-term exposure of rabbit aorta to native LDL can diminish its endothelial-dependent response to the calcium ionophore A23187(36) . In contrast, our results showed that oxidized LDL produced a more profound inhibitory effect on NO synthase activity with most of the reduction occurring within 24 h. The reduction in NO synthase mRNA expression most likely contributes to this exaggerated effect although a direct effect on protein expression and/or activity cannot be ruled out. This finding is consistent with animal studies showing that the inhibitory effects of native and oxidized LDL operate through different mechanisms in attenuating the release of endothelial-derived NO(37) .
Interestingly, the IC values of NO synthase activity
for native and moderately oxidized LDL were fairly comparable
indicating that oxidative modification of the LDL particle rather than
its concentration is more important in determining its inhibitory
effects. Indeed, we found that NO synthase mRNA expression is
negatively regulated by the degree of LDL oxidation as measured by the
formation of malondialdehyde. Changes in the LDL particle associated
with oxidative modification include lipid peroxidation, formation of
conjugated dienes, carbonyl modification of apoB-100, and enzymatic
conversion of phosphatidylcholine to lysophosphatidylcholine by
phospholipase A
in apoB-100(38, 39) .
Recent reports suggest that attenuation of bradykinin-stimulated
calcium transients and inhibition of endothelial-dependent relaxation
by oxidized LDL is mediated by its lysophosphatidylcholine
component(40, 41) . However, studies using different
preparations of human oxidized LDL with similar lysophosphatidylcholine
content demonstrate varying inhibitory effects on endothelium-dependent
relaxations of rabbit aorta(8) . These findings suggest that
components in oxidized LDL other than lysophosphatidylcholine may also
be influencing NO synthase expression and activity. It remains to be
determined what the roles of each of these components in oxidized LDL
are in regulating NO synthase mRNA expression and enzymatic activity.
The effect of oxidized LDL on NO synthase expression in atherosclerosis, however, remains controversial. Studies of human atherosclerotic plaques with in vitro hybridization techniques demonstrate normal expression of NO synthase in the overlying endothelium(42) . An increase in NO production was reportedly found in aorta from cholesterol-fed rabbits although NO activity was greatly reduced as a result of enhanced inactivation(11) . Yet, considerable evidence including the findings in this study suggest that oxidized LDL reduces endothelial NO production at the level of NO synthase(43) . Indeed, we have found a strong correlation between decreases in NO synthase mRNA expression and reduction in NO synthase activity.
Several possible explanations could account for this discrepancy. First, oxidized lipids may affect other cell types such as macrophages and vascular smooth muscle cells which could indirectly influence NO synthase expression in vivo. Second, the regulation of NO synthase protein by oxidized LDL is not known and may differ from that of NO synthase mRNA. Third, oxidized LDL preparations used in various studies or found in atherosclerotic arteries may differ with respect to the concentrations encountered by endothelial cells or the degree of oxidative modification. Perhaps exposure of endothelial cells to lower concentrations of native or mildly-modified LDL would not alter NO synthase mRNA expression while higher levels of oxidative modification would inhibit expression. Finally, NO synthase mRNA transcription and its degradation may be regulated independently by different components of oxidized LDL. For example, certain factors in oxidized LDL which destabilize NO synthase mRNA may either be absent or counteracted by other mediators in the atherosclerotic lesion. Consequently, increases in NO synthase mRNA expression would result from the delayed transcriptional induction by oxidized LDL.
In summary, regulation of endothelial NO synthase expression involves complex interactions between transcriptional and post-transcriptional effects of oxidized LDL. It remains to be determined which component(s) of oxidized LDL and which newly synthesized protein(s) mediate these effects.