© 2000 American Heart Association, Inc.
Vascular Biology |
Apolipoprotein E Inhibition of Vascular Smooth Muscle Cell Proliferation but Not the Inhibition of Migration Is Mediated Through Activation of Inducible Nitric Oxide Synthase
From the Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio.
Correspondence to David Y. Hui, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 231 Bethesda Ave, Cincinnati, OH 45267-0529.
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Abstract |
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Abstract—Initial experiments revealed that low concentrations of apolipoprotein (apo) E (0.1 to 5 µg/mL) were effective in inhibiting platelet-derived growth factor (PDGF)–directed smooth muscle cell (SMC) migration by 60% to 80%. In contrast, higher concentrations of apoE, at 25 and 50 µg/mL, were necessary to achieve similar inhibition of PDGF-induced SMC proliferation. The potential role of nitric oxide (NO) in mediating the inhibitory effects of apoE was explored. Results showed that, although 0.1 to 5 µg/mL of apoE had no effect on NO production by SMCs, physiological concentrations of apoE (25 to 50 µg/mL) enhanced NO synthesis by 2-fold in a dose-dependent manner (P<0.001). Reverse transcription–polymerase chain reaction amplification of RNA obtained from control and apoE-treated SMCs demonstrated a direct role of apoE in activating inducible nitric oxide synthase (iNOS) gene expression. The apoE-induced nitric oxide production was significantly reduced by coincubation of the cells with aminoguanidine or NG-monomethyl-L-arginine (P<0.05) or with antisense iNOS oligodeoxynucleotides (P<0.01). Moreover, the inhibition of iNOS was shown to overcome apoE suppression of PDGF-induced vascular SMC proliferation. However, apoE suppression of PDGF-directed SMC migration was not affected by these treatments. Taken together, these results document that apoE exerts its inhibitory effects on cell proliferation via activation of iNOS. However, apoE inhibition of cell migration is mediated by a mechanism independent of iNOS activation.
Key Words: apolipoprotein E • smooth muscle cell proliferation • nitric oxide • smooth muscle cell migration
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Introduction |
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Apolipoprotein (apo) E is one of the major protein components of plasma lipoprotein in humans and plays an important role in preventing atherosclerotic disease.1 The antiatherogenic property of apoE has been attributed to its ability in mediating lipid clearance from the plasma2 and in promoting cholesterol efflux from peripheral cells for reverse cholesterol transport.3 4 ApoE also possesses antioxidant activity,5 6 suggesting that apoE may also protect against atherosclerosis by limiting lipid oxidation. In addition to these effects on lipid transport and modification, apoE has also been shown to regulate cell functions by modulating signaling pathways in cells that are important for the atherogenic process. Recently, we reported that apoE inhibits platelet-derived growth factor (PDGF)– and oxidized LDL–induced vascular smooth muscle cell (VSMC) migration and proliferation.7 We documented that these effects are mediated by apoE suppression of mitogen-activated protein (MAP) kinase activation and the induction of cyclin D1 gene expression.7 In addition, apoE has been reported to suppress agonist-induced platelet aggregation by activation of nitric oxide (NO) synthesis.8 ApoE also stimulates NO synthesis in human macrophages.9
NO is an important modulator of cell functions, capable of mediating
numerous physiological and
pathophysiological processes in the body. It is
synthesized by 3 isoenzymes of NO synthase (NOS), a constitutively
expressed NOS identified initially in endothelium
(eNOS, or type III NOS), a neuron-specific NOS (nNOS, or type I NOS),
and an inducible form of NOS (iNOS, or type II NOS) that is
activated by cytokines and endotoxins.10
Among these enzymes, iNOS is reported to be found in a number of
different cell types in the vessel wall, including VSMCs after
arterial balloon injury or exposure to inflammatory
cytokines, such as interleukin-1ß, tumor necrosis factor, and
-interferon.11 12 13 14 15 The current literature suggests that
NO in the vessel wall may have beneficial effects against premature
atherosclerosis. Enhancement of vascular NOS activity
by long-term administration of the NO precursor L-arginine
was effective in reducing atherosclerosis in
hypercholesterolemic rabbits16 17 and in
LDL receptor knockout mice.18 Dietary
L-arginine supplementation was also effective in reducing
intimal hyperplasia in rabbits after balloon
angioplasty.19 The relationship between
L-arginine supplementation, NO synthesis, and suppression
of vascular lesions was demonstrated by experiments showing that local
administration of L-arginine to the vessel wall was
effective in enhancing NO generation and inhibiting angioplasty-induced
lesion formation in rabbits.20 Gene transfer of iNOS to
injured arteries has also been shown to improve
endothelium-dependent relaxation and diminish intimal
lesions after balloon angioplasty.21 In contrast,
administration of NOS inhibitors after balloon injury
increased neointimal hyperplasia.22 These in
vivo observations supported the in vitro data showing that NO has
antiproliferative and antimigratory properties on
VSMCs.23 24
In view of our previous report that apoE also inhibits smooth muscle cell proliferation and migration, we tested the hypothesis that the inhibitory properties of apoE on VSMCs are mediated by increased NO production. Results reported here documented that apoE suppresses PDGF-induced SMC proliferation by stimulating iNOS gene expression. Our data also revealed that apoE inhibition of SMC migration is mediated by a discrete mechanism that is independent of iNOS activity.
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Methods |
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Cell Culture
The A7r5 cells obtained from American Type Culture Collection were maintained in DMEM containing 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mmol/L L-glutamine. Cells between passages 3 and 15 were made quiescent by incubation for 48 hours in the presence of 0.4% FBS before use.
Inhibitors of NOS
The NOS inhibitors
NG-monomethyl-L-arginine
(L-NMMA),
NG-monomethyl-D-arginine
(D-NMMA), and aminoguanidine were obtained from Sigma Chemical
Company.
Preparation of Purified Apolipoproteins
Human apoE was isolated from fresh plasma obtained from normal
healthy volunteers as detailed by Rall et al.25 The purity
of each apoE preparation was verified on the basis of a single band
with Mr=34 000 in
SDS-polyacrylamide gels. The purified apoE was stored frozen in
small aliquots and thawed only once before each experiment. The apoE
was added directly to cell culture medium before reconstitution with
lipids. In selected experiments, the arginine residues in apoE were
modified by cyclohexanedione treatment according to the procedure
described previously26 before use. Human apoA-I (a
gift from Dr W. Sean Davidson at our institution) was purified from
plasma HDL.27
Preparation of Oligodeoxynucleotides
Phosphorothioate antisense oligodeoxynucleotide
(5'-CAG-GGGCAAGCCATGTC-3') and mismatched
oligodeoxynucleotide (5'-CACCGCCATGGCATCTG-3') to iNOS were
synthesized on an Applied Biosystems DNA synthesizer on the basis of
the sequence derived from bases -3 to 14 of rat iNOS mRNA, with base 1
being the translation start site.28 The antisense and
mismatched oligodeoxynucleotides were reconstituted in PBS
just before their administration to cells.
SMC Proliferation
The proliferation of A7r5 SMCs was determined on the basis of
[3H]thymidine incorporation into cellular DNA
as described previously.7 Briefly, quiescent A7r5 cells
were plated in 96-well tissue culture dishes at a density of
5x103 cells/well and then incubated for 24 hours
at 37°C with media containing the indicated concentrations of apoE,
PDGF-BB (Life Technologies), aminoguanidine, and
oligodeoxynucleotides. In the antisense experiments,
serum-free medium was used to prevent degradation of
oligonucleotides by nuclease in the serum. For
determination of [3H]thymidine incorporation
into cellular DNA, 1 µCi of [3H]thymidine was
added to the cell culture media 5 hours before the end of the
experiment. Cells were washed twice with PBS, followed by incubation at
4°C in 25% trichloroacetic acid for 20 minutes. The plates were then
washed 3 times with cold 25% trichloroacetic acid, followed by the
addition of 0.25 mol/L NaOH. Radioactivity in the cell lysate was
quantified by liquid scintillation counting.
SMC Migration
The migration of A7r5 toward a PDGF gradient was examined
according to the procedure of Law et al,29 as described
previously.7 Briefly, quiescent A7r5 cells were suspended
in DMEM containing 0.4% FBS at a concentration of
2x105 cells/mL. The cells were incubated in
solution with or without apoE for 30 minutes at 37°C, and 0.1-mL
aliquots were added to the top chamber of tissue culture–treated
Transwell polycarbonate membrane with 8-µm pores in 24-well
plates (Corning Costar). The lower Transwell compartment contained
0.6 mL of DMEM, 0.4% FBS, with or without 10 ng/mL PDGF-BB. After a
4-hour incubation period at 37°C, the upper surfaces of the filters
were washed with PBS, fixed with methanol for 10 minutes at 4°C, and
then stained with hematoxylin. The number of SMCs that migrated to the
lower surface of the filters was determined microscopically by counting
in different high-power fields at a magnification of x320.
NOS Activity Assay
NOS activity was determined by the conversion of
[3H]arginine to
[3H]citrulline by use of the NOSdetect assay
kit from Stratagene, according to the modified method of Riddell et
al.8 Briefly, 1.5x105 cells in
6-well plates were lysed in 200 µL of lysis buffer containing 25
mmol/L HEPES (pH 7.5), 0.2 mmol/L PMSF, 0.05% 2-mercaptoethanol,
and 1% Triton X-100. The protein concentration of the cell lysate was
adjusted to 10 mg/mL, and 10 µL of the cell lysate was then incubated
for 1 hour at 37°C with 40 µL of substrate buffer containing
50 mmol/L Tris-HCl, pH 7.4, 1 mmol/L NADPH, 6 µmol/L
tetrahydrobiopterin, 2 µmol/L flavin adenine
dinucleotide, 2 µmol/L flavin adenine
mononucleotide, 1.2 mmol/L
CaCl2, and 1 µCi
[3H]arginine. The reaction was terminated by
addition of 400 µL of 50 mmol/L HEPES (pH 5.5), 5 mmol/L
EDTA, and 100 µL of an equilibrated resin from Stratagene. The
mixture was transferred to a spin filter and microcentrifuged
for 30 seconds at 12 000 rpm. The amount of
[3H]citrulline in the eluate was measured by
liquid scintillation counting. Enzyme-specific reaction was determined
as the total counts minus the mean value of counts in buffer.
NO output was also assessed by measurement of its stable end-product nitrite in the culture medium. The A7r5 cells were seeded into 24-well plates at a density of 2x105 cells/well. Cultured medium after the cells had been exposed to various treatments was collected and then incubated with Griess reagent for 10 minutes. The reaction product was quantified on the basis of absorbance at 550 nm.30
Reverse Transcription–Polymerase Chain Reaction
Total RNA was prepared from A7r5 cells by the guanidine
thiocyanate–phenol-chloroform method.31 Total RNA (2
µg) from each sample was reverse-transcribed into cDNA by SuperScript
II reverse transcriptase (Life Technologies). Polymerase chain reaction
(PCR) amplification of the iNOS cDNA was done with
oligonucleotide primers designed according to the
published sequence of rat iNOS.28 The forward primer
sequence was 5'-GGAAAGTC-GGAAGCGCTAGCC-3', and the reverse primer
sequence was 5'-GGTGAACACGTTCTTGGCGTGG-3'. The reaction was conducted
with an initial denaturation step at 94°C for 3 minutes, followed by
38 cycles each consisting of incubation at 94°C for 1 minute, 58°C
for 75 seconds, and 72°C for 90 seconds. The size of the PCR
product was assessed by electrophoretic migration on 1.5% agarose
gels. The expression level of iNOS was semiquantified on the basis of
kinetic analysis of reverse transcription (RT)-PCR with GAPDH
mRNA as the standard, as described.32 33
Statistical Analysis
Two-factorial ANOVA was performed to estimate the difference
between the apoE effects on cell migration and proliferation. In other
experiments, 1-way ANOVA was performed to determine significant
difference between 2 data in different conditions. A value of
P<0.05 was considered to be statistically significant.
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Results |
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ApoE Inhibition of PDGF-Induced SMC Migration and Proliferation
Previous studies with rat A7r5 embryonic SMCs and human coronary artery SMCs have demonstrated that physiological concentrations of apoE (25 to 50 µg/mL) strongly inhibited PDGF- and oxidized LDL–induced cell migration and proliferation.7 Using the A7r5 cells as the model, the present study showed that lower concentrations of apoE (0.1 to 5 µg/mL) were sufficient in inhibiting PDGF-directed SMC migration by 60% to 80% (Figure 1
). In
contrast, these low levels of apoE were significantly less effective in
inhibiting PDGF-induced SMC proliferation, with only 30% inhibition
observed at 5 µg/mL (Figure 1). These findings suggested the
possibility that apoE may exert its inhibitory effects on
VSMC migration and proliferation by discrete mechanisms. The
specificity for apoE inhibition of SMC migration and proliferation was
verified by experiments showing that apoA-I, at 50 µg/mL, had no
effect on SMC response to PDGF (data not shown). In addition,
cyclohexanedione modification of the arginine residues in apoE, which
was shown to inhibit apoE interaction with receptors,26
also abolished its inhibition of PDGF-stimulated SMC migration and
proliferation. Similar results were also observed with human
arterial SMCs and with primary mouse aortic SMCs.
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), quiescent A7r5
cells were incubated for 30 minutes at 37°C with the indicated
concentrations of apoE before their addition to Transwell membranes
in 24-well plates at a density of 2x104 cells/well. The
number of cells that migrated toward 10 ng/mL PDGF in the lower chamber
was determined microscopically by counting in different high-power
fields at a magnification of x320. In both sets of experiments, 100%
maximum response represents data obtained from cells incubated
with PDGF in the absence of apoE after the background values observed
in the absence of PDGF stimulation had been subtracted. The data
represent the mean±SD from 5 different experiments with 2
different preparations of apoE. *P<0.05 vs 100%
maximum value; **P<0.001 vs 100% maximum value;
P<0.001, significant difference between the response
curves.
ApoE Enhancement of NO Production in SMCs
The possible involvement of NO in mediating apoE inhibition of SMC
migration and/or proliferation was explored by determination of NO
activity in cells incubated with or without apoE. Results showed that
lysates prepared from cells incubated with
physiological concentrations (50 µg/mL) of apoE
displayed a 2-fold increase in NOS in comparison with cells incubated
without apoE (Figure 2).
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Apolipoprotein E stimulation of NOS activity in SMCs was confirmed
directly by assessing NO production on the basis of the
presence of its stable end-product nitrite in the cell culture
medium. Results, as depicted in Figure 3, showed that whereas 0.1 to 5 µg/mL of apoE had no stimulatory effect
on NO production, incubation of SMCs with 25 to 50 µg/mL of
apoE for 24 hours significantly enhanced NO production by
2-fold in a dose-dependent manner (P<0.001). The
apoE-stimulated NO production was inhibited by aminoguanidine
(Figure 3), a selective inhibitor of inducible
NOS,34 35 or preincubation of cells with 10
µmol/L antisense oligodeoxynucleotides against iNOS mRNA
(Figure 4).
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ApoE Induction of Inducible NOS Gene Expression in VSMCs
A direct effect of apoE on iNOS expression in SMCs was examined by
RT-PCR amplification of cellular RNA with iNOS-specific
oligonucleotide primers. Under basal unstimulated
conditions, iNOS mRNA was not detectable in the VSMCs even by the
sensitive RT-PCR procedure (Figure 5). In
contrast, RT-PCR of RNA isolated from cells incubated for 6 hours with
25 and 50 µg/mL of apoE resulted in the amplification of a
product with the expected size of 561 bp (Figure 5). The
authenticity of the RT-PCR product as iNOS cDNA was confirmed on
the basis of restriction digestion with XbaI and
PvuII, which yielded the expected cDNA fragments of 510 and
371 bp, respectively (data not shown). The inability to detect iNOS
mRNA in control SMCs was not due to differences in the amount of RNA in
the samples, because kinetic analysis of GAPDH mRNA expression
demonstrated that equal amounts of reverse-transcribed DNA were applied
to each PCR reaction (data not shown). Thus, the NOS activity observed
in quiescent SMCs (Figure 2
) was most likely mediated by a
different isoform of NOS. In this regard, a recent report demonstrated
the presence of neuronal NOS in rat VSMCs.36
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NO-Mediated ApoE Suppression of PDGF-Induced SMC
Proliferation
The observation that incubation of SMCs with 25 and 50 µg/mL
apoE resulted in iNOS activation suggested that apoE suppression of
PDGF-induced cell proliferation may be mediated through the NO pathway.
This hypothesis was examined directly by determination of the impact of
the NOS inhibitor aminoguanidine and L-NMMA on apoE
suppression of PDGF-induced SMC responses. The results showed that
although apoE inhibited PDGF-induced
[3H]thymidine incorporation into cellular DNA
(P<0.05), 0.5 mmol/L aminoguanidine was effective in
alleviating apoE suppression of PDGF-induced DNA synthesis
(P<0.05) (Figure 6).
Furthermore, the active form of NMMA, L-NMMA, had a reversal effect on
apoE inhibition of PDGF-induced SMC proliferation, but the inactive
form D-NMMA had no effect. These results suggest that apoE inhibition
of cell proliferation is mediated by NOS activity.
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Next, we used an antisense oligonucleotide strategy to
test the hypothesis that apoE stimulation of NO synthesis is due to
activation of iNOS, rather than eNOS or nNOS. Results showed that apoE
inhibition of PDGF-induced cell proliferation was abolished by
antisense iNOS oligodeoxynucleotides in a
concentration-dependent manner (Figure 7). In contrast, mismatched
oligodeoxynucleotides had no effect on apoE suppression of
PDGF-induced SMC proliferation (Figure 7). Taken together, these
experiments documented a direct role of apoE-stimulated iNOS activity
on the suppression of PDGF-induced cell growth.
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ApoE Suppression of PDGF-Directed SMC Migration Is Independent of
NO Production
The observation that iNOS activation required 25 to 50 µg/mL of
apoE, yet PDGF-directed SMC migration could be inhibited by apoE at 0.1
to 5 µg/mL, suggested the possibility that apoE inhibition of cell
migration is independent of NOS activation. Indeed, neither 0.1 or
0.5 mmol/L aminoguanidine was capable of reducing the potency of
apoE in inhibiting PDGF-directed SMC migration (data not shown).
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Discussion |
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The results of the present study provided strong evidence that apoE inhibits PDGF-induced VSMC migration and proliferation through discrete mechanisms. Moreover, the pathway for apoE inhibition of SMC proliferation was shown to be mediated by stimulation of NO production via iNOS induction. These observations are consistent with our previous results that documented the role of apoE in regulating signal transduction pathways leading to the inhibition of SMC growth. Our previous study showed that apoE inhibits PDGF-induced SMC proliferation by inhibiting growth factor–mediated MAP kinase activation and retaining cells at the G0 phase of the cell cycle.7 NO has been shown previously to inhibit SMC growth by arresting cells at the S phase, followed by a shift back in the cell cycle from the G1-S boundary to the quiescent G0 state.37 Because MAP kinase activation can be inhibited by NO,38 39 because of its suppression of Ras-dependent Raf-1 activation,38 it is quite likely that apoE inhibition of MAP kinase activation and SMC proliferation is a direct result of its ability to activate iNOS gene expression. Results showing that aminoguanidine, L-NMMA, and antisense iNOS oligonucleotide inhibition of iNOS activity could effectively ameliorate apoE inhibition of PDGF-induced SMC proliferation are supportive of this hypothesis.
The results of the present study also showed that apoE inhibits SMC
migration by a pathway that is independent of iNOS activation. The
discrete mechanism by which apoE inhibits PDGF-induced SMC
proliferation and migration is consistent with previous reports
of distinct cell signaling pathways mediating these
events.40 41 Bornfeldt et al40 demonstrated
that PDGF-induced SMC proliferation proceeds through a MAP kinase
kinase– and MAP kinase–modulated mechanism, whereas PDGF-directed SMC
migration was shown to coincide with stimulation of
phosphatidylinositol turnover, diacylglycerol formation, and
intracellular calcium ion flux. Apo E stimulation of NO
production (Figures 2 and 3) and the ability of
NO to inhibit MAP kinase activation38 39 suggested a
direct effect of apoE in inhibiting the MAP kinase signaling cascade
required for cell proliferation, as discussed earlier. The ability of
apoE to also inhibit SMC migration at a concentration that did not
activate iNOS suggested that apoE may also interfere with the
phosphatidylinositol signaling cascade required for cell migration.
The mechanisms by which apoE inhibits the separate signaling cascades required for growth factor–induced SMC proliferation and migration are yet to be established. However, the obliteration of its inhibitory effects by cyclohexanedione modification suggested that apoE binding to specific receptors on the surface of the SMCs is a prerequisite. Currently, apoE receptors reported to be expressed on the SMC surface include the LDL receptor, the LDL receptor–related protein (LRP), the VLDL receptor, apoE receptor-2, and heparan sulfate proteoglycans.42 43 44 It is unlikely that apoE regulation of SMC response is mediated through the LDL receptor or the apoE receptor-2. This conclusion is based on the observation that apoE was effective in inhibiting PDGF-induced SMC response before its reconstitution with lipids to form a lipoprotein complex. Previous studies have documented that lipid-free apoE does not interact with LDL receptor or with apoE receptor-2.45 46 Although the lipid-free apoE may have complexed with lipoproteins present in the serum in the cell culture media, this possibility is unlikely, for 2 reasons. First, the LDL receptor was not required for apoE inhibition of lymphocyte response to mitogenic activation.47 48 Second, lipid-free apoE was unable to inhibit ADP-induced platelet aggregation even in the presence of plasma.8 Because apoE inhibition of platelet aggregation was recently shown to be mediated through apoE receptor-2,46 the apoE receptor-2–mediated signal transduction pathway requires formation of an apoE-lipid complex even when the experiments were performed in the presence of plasma or serum.
Recent reports have documented the interaction of LRP, VLDL receptor, and apoE receptor-2 with cytosolic signaling adaptor proteins in neuronal cells.49 50 The LRP has also been shown to interact with GTP-binding proteins in melanocytes and liver cells.51 These results suggested the possibility that these receptors may have signal transduction capabilities in these cell types. Interestingly, LRP has been reported to be involved in signal transduction events in macrophages and brain cells.52 53 54 It is particularly important to note that the LRP-mediated cell-signaling events can occur with lipid-free apoE without its formation of an apoE-lipid complex.53 54 Accordingly, it is possible that LRP may be responsible for mediating the currently observed apoE effects on SMCs. The previous observations that 2 different LRP ligands, namely anti-LRP antibody and recombinant receptor-associated protein, were effective in inhibiting VSMC migration support this hypothesis.55 56 However, ligand binding to cell surface heparan sulfate proteoglycans has also been shown to induce signaling events leading to suppression of SMC migration.57 Thus, it is possible that apoE inhibition of PDGF-directed SMC migration may be mediated by its interaction with LRP and/or through its interaction with cell surface heparan sulfate proteoglycans. Likewise, apoE inhibition of PDGF-induced SMC proliferation may also be mediated by signaling events subsequent to its interaction with other apoE receptors or cell surface heparan sulfate proteoglycans. In this regard, the possibility has been suggested that apoE interaction with heparan sulfate proteoglycans interferes with basic fibroblast growth factor signaling through its cognate receptor.58 The precise role of LRP, heparan sulfate proteoglycans, and other apoE receptors in mediating the vascular cytostatic function of apoE remains to be defined.
Regardless of the receptors that mediate apoE inhibition of SMC migration and proliferation, the data reported here demonstrated that apoE may protect against vascular diseases via activation of iNOS in SMCs. Fukumoto et al59 showed that iNOS generation of NO protects the vasculature against cytokine-induced inflammatory coronary lesions. Thus, the present study suggests that apoE gene transfer may be a viable therapeutic option for suppressing inflammatory response to arterial injury, such as those observed in restenosis after balloon angioplasty.
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Acknowledgments |
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This research was supported in part by funds from the National Institutes of Health (grant HL-61332) and the Japan Research Foundation for Clinical Pharmacology. Dr Swertfeger was the recipient of a Postdoctoral Fellowship (9920615V) from the Southern and Ohio Valley Consortium of the American Heart Association.
Received July 6, 1999; accepted November 19, 1999.
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