© 1996 American Heart Association, Inc.
Articles |
Active Oxygen Species and Lysophosphatidylcholine Are Involved in Oxidized Low Density Lipoprotein Activation of Smooth Muscle Cell DNA Synthesis
From the Division of Cardiology, Cedars-Sinai Medical Center, Los Angeles, Calif (A.S., J.R., P.K.S., B.C.), and King Gustaf V Research Institute, Karolinska Institute, Stockholm, Sweden (A.S. J.R., J.N.).
Correspondence to Jan Nilsson, King Gustaf V Research Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden.
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Abstract |
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Abstract It has recently been shown that oxidative modification of LDL enhances the mitogenic effect of LDL on smooth muscle cell (SMC) DNA synthesis. However, because of its complex chemical structure, the mitogenic components have not been well characterized. Exposure of LDL to the oxidant Cu2+ is followed by a rapid accumulation of peroxides that peaks after 8 to 12 hours and a conversion of the phospholipid phosphatidylcholine into lysophosphatidylcholine that continues for up to 48 hours. Most of the mitogenic activity is formed during the first 4 hours of oxidation. Both superoxide dismutase and catalase effectively inhibit the mitogenic activity of oxidized LDL, suggesting involvement of reactive oxygen intermediates. In the presence of 1% serum, low concentrations of hydrogen peroxide activated SMC DNA synthesis in a dose-dependent manner, with a maximal effect at a concentration of 200 µmol/L, whereas higher concentrations were inhibitory. Lysophosphatidylcholine also enhanced SMC DNA synthesis, with a maximal stimulation at a concentration of 10 µmol/L. Oxysterols, which also accumulate in oxidized LDL, effectively inhibited DNA synthesis. These results demonstrate that oxidation of LDL is associated with formation of several substances affecting the growth of SMCs. Among these substances, low levels of reactive oxygen intermediates and lysophosphatidylcholine stimulate DNA synthesis, whereas at a higher concentration they, as well as oxysterols, are inhibitory.
Key Words: atherosclerosis • lipid oxidation 
phospholipids • cell proliferation
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Introduction |
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Hypercholesterolemia is a major risk factor for development of atherosclerosis in humans.1 Studies in experimental animals have shown that hypercholesterolemia is associated not only with formation of fatty lesions but also with induction of intimal SMC proliferation and formation of fibromuscular plaques.2 The mechanisms by which hypercholesterolemia promotes SMC proliferation in atherosclerosis are poorly understood. In experimental animals, hypercholesterolemia induces a vascular inflammatory reaction followed by development of intimal fibrosis caused by infiltrating medial SMCs.3 Over the last few years evidence suggesting that oxidative modification of LDL plays a key role in this process has accumulated.4 5 Experimental studies have demonstrated that oxidized LDL acts as a proinflammatory agent by stimulating the release of leukocyte chemoattractants from endothelial cells,6 activating endothelial expression of leukocyte cell adhesion molecules,7 promoting differentiation of monocytes into resident macrophages,8 and playing a critical role in the formation of foam cells.9
Hypercholesterolemia and lipid oxidation have also been implicated in activation of vascular SMC growth by more direct mechanisms. In culture, SMCs proliferate more rapidly in serum isolated from hypercholesterolemic patients than in serum from normocholesterolemic individuals.10 Exposure of SMCs to isolated LDL activates some mitogenic signal pathways and potentiates the effect of other growth factors.11 12 More recently, it has been shown that oxidized LDL is more potent in this respect than native LDL. Exposure of human SMCs to oxidized LDL increases PDGF-A chain and PDGF receptor expression, indicating possible mechanisms for both autocrine activation of cell growth and increased sensitivity to exogenous growth factors.13 Experiments demonstrating that antioxidant treatment inhibits the development of neointimal thickening after balloon injury of the aorta in hypercholesterolemic rabbits14 15 have provided evidence for a direct role of lipid oxidation in also regulating SMC proliferation in vivo.
Previous observations that the mitogenic activity of oxidized LDL is inhibited by superoxide dismutase is indicative of an involvement of ROIs.13 However, because of its complex chemical structure it has been difficult to precisely determine the mitogenic components of oxidized LDL. Therefore, the present study was undertaken to identify the components of oxidized LDL responsible for stimulation of DNA synthesis in vascular SMCs.
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Methods |
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Materials
All media and sera were from GIBCO. Xanthine, xanthine oxidase, superoxide dismutase, catalase, phosphatidylcholine (dipalmitoyl), lysophosphatidylcholine (palmitoyl), phosphatidylserine (dipalmitoyl), lysophosphatidylserine (stearic acid), phosphatidic acid (dipalmitoyl), sphingomyelin (mostly stearic and nervonic acid), and sphingosine were purchased from Sigma. An
-actin–specific antibody (HHF 35) was kindly provided by Dr
A. Gown, University of Washington, and purified PDGF-AA by Dr C.-H.
Heldin, University of Uppsala, Sweden. 5,6-Epoxide,
cholestane-3ß,5,6ß-triol,
7-hydroxycholesterol,
7ß-hydroxycholesterol,
7-ketocholesterol,
24-hydroxycholesterol,
25-hydroxycholesterol, and
27-hydroxycholesterol were gifts from Dr I.
Björkhem, Karolinska Institute, Stockholm, Sweden.
Isolation and Culture of SMCs
SMCs were isolated from normal
aortic media obtained from a
transplantation donor and from the media of a saphenous vein graft.
Tissue specimens were carefully dissected, cut into small pieces, and
allowed to attach to the surface of six-well multiplates by drying
for 15 minutes. The explants were then cultivated in DMEM/F-12
containing 10% NCS and 50 µg/mL gentamycin at 37°C in an
atmosphere of 5% CO2 in air. Cells began migrating out
from the explants within 1 to 2 weeks and reached confluence within
another 2 weeks. Secondary cultures were established by trypsinization
and seeding of the cells in 75-cm2 culture flasks. Rat SMCs
were isolated by enzymatic technique as described
earlier.16 The purity of the SMC populations was
determined by the presence of muscle-specific -actin
immunoreactivity by using the HHF 35 antibody.17 Cells in
the 8th to 15th passages were used in the experiments. All three SMC
populations (ie, rat aortic, human artery, and human vein SMCs) used in
the present study showed equal response to stimulation with LDL and
oxidized LDL. Experiments analyzing the effect of hydrogen peroxide and
phospholipids were performed on human SMCs, and the results were
verified in at least two independent experiments with both artery and
vein SMCs.
Analysis of DNA Synthesis
Subconfluent cultures of SMCs grown
on 22x22-mm glass
coverslips in six-well plates were growth arrested by transfer to
serum-free F-12 medium/0.1% BSA for 48 hours. The cells were then
incubated in experimental medium containing [3H]thymidine
to a final concentration of 2 µCi/mL for 48 hours or indicated time
intervals and fixed in 3% glutaraldehyde with 0.1
mol/L sodium cacodylate. The specimens were dehydrated in ethanol and
mounted on glass slides. The slides were dipped in Kodak NTB2 emulsion,
air dried, and exposed at 4°C for 3 days. They were subsequently
developed in Kodak D-19, stained in methylene blue, and the fraction of
labeled nuclei determined by counting of at least 300 randomly selected
cells on each coverslip. Phospholipids and oxysterols used for
analysis of DNA synthesis were dissolved in ethanol. The final
concentration of ethanol in the test sample was 0.5% or less. Control
experiments demonstrated that this concentration of ethanol did not
affect the rate of DNA synthesis.
Preparation of Native and Oxidized LDL
Venous blood was drawn
into precooled vacutainer tubes
containing Na2 EDTA (1.4 mg/mL). Plasma was then recovered
by centrifugation at 1400g for 20 minutes at
+1°C. The isolated plasma was adjusted to a density of 1.10 kg/L by
addition of NaCl. A density gradient consisting of 3 mL of
1.10-kg/L-density plasma and 3 mL of 1.065-, 1.020-, and 1.006-kg/L
NaCl solutions, respectively, was then formed in cellulose nitrate
tubes (Ultraclear tubes, Beckman) and centrifuged (Beckman
L8-55 ultracentrifuge, 40 000 rpm) in a Beckman SW 40
swinging-bucket rotor at 1°C overnight.18 The VLDL
and IDL fractions were aspirated from the top 3 mL, and LDL was
harvested from the next 4 mL of the tube. The EDTA was subsequently
removed by filtration on a Sephadex PD-10 column (Pharmacia). The
protein content of the LDL preparation was determined as described by
Lowry et al.19 LDL was oxidized by incubation in 5.0
µmol/L CuSO4/PBS for 18 hours or indicated time
intervals at 37°C. The copper was removed by filtration on a Sephadex
PD-10 column. The oxidized LDL was diluted and used immediately without
addition of antioxidants.
Analysis of LDL Oxidation Products
Peroxide levels were
analyzed using determination of the
peroxide-dependent conversion of iodide to iodine as described by
El-Saadani et al.20 Aldehydes were determined by the TBARS
assay and expressed as malondialdehyde equivalents.21 The
extent of oxidative modification of LDL was also determined by agarose
gel electrophoresis.
Analysis of LDL Phospholipids
Ten milliliters of LDL (200
µg/mL) was exposed to copper for
various times up to 48 hours. The oxidation was terminated by addition
of 24 µmol/L EDTA and 20 µmol/L BHT. LDL was then concentrated
fivefold by centrifugation in a density gradient as
described above. The reisolated LDL was desalted by gel filtration on a
Sephadex PD-10 column. Lipids in 0.5 mL of LDL were extracted by
addition of 2.5 mL of methanol and 5.0 mL of chloroform. After
evaporation, the extracted lipids were resolved in 50 µL of
chloroform/methanol (1:1). Phospholipids were separated by
thin-layer chromatography in a solvent of
chloroform/methanol/water (69:35:6), using isolated phospholipids as
reference. Phospholipids were visualized by exposure to iodine, and the
lysophosphatidylcholine, sphingomyelin, and phosphatidylcholine bands
were removed by scraping. They were then combusted to free phosphor by
heating at 200°C for 5 hours and quantified as described by
Bartlett.22
Statistical Methods
Values are given as mean±SD.
Between-group analyses
were made with an unpaired t test. A value of
P<.05 was considered significant.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Changes in LDL Composition During Oxidation
Peroxide levels increased rapidly during the first hours of oxidation, reaching a peak of
1 µmol/mg LDL protein after 8 to 12
hours (Fig 1
). At 24 hours, peroxide levels remained
elevated but had returned to baseline values after 44 hours. LDL
aldehyde levels, determined as the content of TBARS, increased
continuously during the 48 hours studied (Fig 1). The major
phospholipids found in native LDL were phosphatidylcholine (1013
nmol/mg LDL protein), sphingomyelin (564 nmol/mg LDL protein), and
lysophosphatidylcholine (33 nmol/mg LDL protein), which together
accounted for more than 90% of the phospholipids in LDL (Fig
2). After 9 hours' exposure to Cu2+,
about 10% of the phosphatidylcholine had been hydrolyzed into
lysophosphatidylcholine, resulting in a fivefold increase in the
lysophosphatidylcholine content (Fig 2). At 48 hours, the
lysophosphatidylcholine content of LDL was 497 nmol/mg LDL protein, and
the phosphatidylcholine content had been reduced to 601 nmol/mg LDL
protein. The sphingomyelin content of LDL did not change significantly
during oxidation (Fig 2). An increased agarose mobility was
observed
after 4 hours' exposure to copper, and a maximum mobility was
present after 24 hours (data not shown).
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), sphingomyelin (
), and
lysophosphatidylcholine (
Effect of Oxidation on Mitogenic Activity
Exposure of SMCs to
native or oxidized LDL in serum-free
medium did not result in stimulation of DNA synthesis (data not shown).
In the presence of 1% NCS, 0.5 µg/mL of oxidized LDL increased DNA
synthesis from 33.0±1.7% to 42.7±3.2% (P<.05). At a
concentration of 5 µg/mL of oxidized LDL, 57.0±2.0% of the cells
replicated their DNA (P<.005 versus the control containing
only 1% NCS), whereas higher concentrations resulted in inhibition of
DNA synthesis (Fig 3). Addition of 5 µg/mL of LDL was
also found to increase SMC DNA synthesis, but it was less effective
than the oxidized LDL preparation (46.5±3.5% versus 57.0±2.0%,
P<.05). LDL had no toxic effects in the concentrations
analyzed in the present study.
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To analyze the time kinetics of the
formation of
growth-promoting factors during oxidation of LDL, 200 µg/mL of
LDL was exposed to 5.0 µmol/L CuSO4. After 2, 4, 8, 24,
and 48 hours, the copper was removed by filtration on a Sephadex PD-10
column. The lipoproteins were then diluted to final concentrations of
10, 20, and 50 µg/mL and incubated with the SMCs for another 48
hours. In this experiment, the fractions of labeled nuclei in control
cells incubated with the respective concentration of freshly isolated
native LDL were 23.0±1.0, 23.3±2.1, and 25.5±0.5%. By
using the
highest concentration of oxidized LDL, an increased
mitogenic activity could be detected after only 2 hours of
oxidation (36.0±4.6% labeled nuclei, P<.05). At this
concentration, a maximal growth-stimulatory activity was observed
after 4 hours of oxidation (44.7±1.5% labeled nuclei,
P<.005 versus 50 µg/mL of native LDL), whereas a
prolonged oxidation resulted in toxic cell death (Fig 4A). At
lower concentrations, increased levels of
mitogenic activity were observed for up to 8 hours of
oxidation. At a concentration of 20 µg/mL, further oxidation resulted
in cytotoxicity. However, at a concentration of 10 µg/mL, the
growth-stimulatory effect remained unchanged after the first 8
hours of oxidation, with no visible signs of cytotoxicity (Fig
4A).
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In a parallel experiment, the effect of storage of
native LDL on
formation of mitogenic activity was analyzed. In
this experiment, LDL was desalted on a Sephadex PD-10 column and stored
in darkness under nitrogen at 4°C for up to 48 hours. No
mitogenic activity could be demonstrated in LDL used
immediately after recovery from the column. However, after 48 hours of
storage, 50 µg/mL of native LDL contained approximately the same
amount of mitogenic activity as 10 µg/mL of LDL oxidized
by copper for 8 hours (Fig 4B
). This effect was not associated
with
detectable changes in peroxides, aldehydes, and lysophosphatidylcholine
(data not shown).
Effect of ROIs on SMC DNA Synthesis
As shown in Fig
1, oxidation of LDL is associated with a
significant accumulation of peroxides. Addition of hydrogen peroxide
stimulated DNA synthesis of SMCs grown in the presence of 1% NCS in a
dose-dependent manner. A maximal stimulation was observed at a
concentration of 200 µmol/L (74.7±6.7% labeled nuclei versus
43.0±5.0% in cells incubated with 1% NCS, P<.005),
whereas toxic effects were observed at higher concentrations (Fig
5). Similar results were obtained in cells grown in the
presence of 5 ng/mL of PDGF-AA, whereas no stimulatory effect was
observed in medium without serum or growth factors. Addition of
superoxide dismutase and catalase also completely inhibited the
mitogenic activity of oxidized LDL (Fig 6).
Addition of superoxide dismutase and catalase in the absence of
oxidized LDL did not affect SMC DNA synthesis (data not shown).
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Effects of Phospholipids on SMC DNA Synthesis
Phosphatidylcholine and sphingomyelin are the major phospholipids
present in native LDL (Fig 2). At a concentration of 10
µmol/L,
these phospholipids did not affect SMC DNA synthesis (Fig 7).
Also, less abundant phospholipids, such as
phosphatidylserine,
lysophosphatidylserine, and phosphatidic acid,
as well as sphingosine, were without significant effect at this
concentration. In contrast, lysophosphatidylcholine increased DNA
synthesis from 31.7±4.2% to 50.7±8.4% (P<.01).
Dose-response experiments demonstrated that a maximum effect on DNA
synthesis was obtained at a lysophosphatidylcholine concentration of 10
µmol/L (Fig 8). A less pronounced stimulation was
obtained in serum-free medium containing 5 ng/mL of PDGF-AA,
whereas no significant growth-stimulatory effect was seen in medium
without serum or growth factors. Addition of 4 IU/mL phospholipase
A2 (an enzyme that converts phosphatidylcholine to
lysophosphatidylcholine) to medium containing 5 µg/mL of native LDL
increased the fraction of labeled nuclei from 42.5±4.2% to
59.8±4.8% (P<.005), whereas phospholipase A2
was without effect on DNA synthesis in the absence of LDL.
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Effect of Oxysterols on SMC DNA Synthesis
Oxidation of LDL is
associated with increased accumulation
of a number of oxysterols, such as 5,6-epoxy
cholesterol, cholestane-3ß,5,6ß-triol,
7-hydroxycholesterol, 7ß-hydroxycholesterol,
7-ketocholesterol, 24-hydroxycholesterol,
25-hydroxycholesterol, and 27-hydroxycholesterol. With the
exception of 25-hydroxycholesterol, all these oxysterols were
found to be potent inhibitors of PDGF-induced DNA synthesis
at a concentration of 1 µg/mL (Fig 9A). At this
concentration, no change in cell morphology was observed and there was
no increase in trypan blue uptake, suggesting that this inhibition was
due to mechanisms other than general cytotoxicity. Oxysterols also
inhibited DNA synthesis in the presence of 1% NCS (Fig 9B), but
this
effect was less pronounced than that observed in serum-free medium
with PDGF-AA.
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Discussion |
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These findings confirm and extend earlier observations that oxidized LDL contains factors that stimulate SMC DNA synthesis.13 These factors are not in themselves sufficient to induce activation of DNA synthesis but act in concert with other growth factors, such as PDGF-AA and other serum mitogens. Most of the mitogenic activity that has accumulated in oxidized LDL after 48 hours is being formed during the first 4 hours after exposure to copper. During this period, there is a significant accumulation of peroxides and presumably also other ROIs in LDL, whereas the lysophosphatidylcholine level still remains relatively low. After 24 hours of oxidative stress, LDL contains large amounts of lysophosphatidylcholine but only small amounts of ROIs. The results of the present study suggest that the mitogenic activity of oxidized LDL is related to the generation of both of these groups of substances. However, when comparing the chemical composition of oxidized LDL with its ability to induce DNA synthesis during a 48-hour exposure to cultured SMCs, one should keep in mind that additional compositional changes may occur during this time. As demonstrated previously,13 the induction of DNA synthesis by native LDL can be explained at least partly by an oxidative modification during preparation or in the cell culture environment.
The notion that ROIs generated during oxidation of LDL are involved in activation of DNA synthesis is also supported by the observation that this effect is inhibited by superoxide dismutase and catalase. Notably, the mitogenic effect of LDL oxidized for 24 hours, a time at which only small amounts of peroxides were detected in LDL, was also inhibited by the antioxidative enzymes, suggesting involvement of ROIs not detected by this assay. The maximum level of peroxides observed in LDL during oxidative modification was 1 nmol/mg LDL protein. The peroxide concentration of medium containing 5 µg/mL of this oxidized LDL should be 5 µmol/L, which is about 10-fold lower than the levels observed to stimulate DNA synthesis. It is, however, possible that local interactions between oxidized LDL and cells will result in exposure to peroxide levels markedly higher than those present in the rest of the medium. Storage of native LDL under nitrogen for 48 hours at 4°C in darkness was associated with formation of about 20% of the mitogenic activity formed in LDL exposed to copper for 8 hours at 37°C. However, the accumulation of this activity was not associated with detectable changes in peroxide, aldehyde, and lysophosphatidylcholine levels. This observation suggests the possibility that the mitogenic activity may be related to the formation of factors other than peroxides and lysophosphatidylcholine, but the results may also be due to peroxide, aldehyde, and lysophosphatidylcholine assay insensitivity.
Recent studies have demonstrated that oxygen radicals may affect
several of the mechanisms involved in regulation of cell growth. Like
most growth factors, they induce intracellular
alkalinization23 and activate transcription of
early-response genes.24 There is also evidence that
intracellular oxygen radicals play a direct role in regulation of gene
transcription. A peroxide-activated transcription factor,
NF-
B, has been identified in eukaryotic
cells.25 After activation, this factor induces
transcription of a number of genes involved in inflammatory and immune
responses, including the SMC mitogen TNF-.26 However,
whether an NF-B–mediated activation of TNF- production
is involved in stimulation of SMC DNA synthesis by oxidized LDL and
hydrogen peroxide remains to be clarified.
The phospholipid lysophosphatidylcholine was also identified as a
mitogen for vascular SMCs. The lysophosphatidylcholine content of
medium containing 5 µg/mL of oxidized LDL is 1 to 2 µmol/L,
which is close to the levels found to activate SMC DNA
synthesis. However, as discussed above, it is likely that higher levels
are present in the local environment of the cells.
Lysophosphatidylcholine is formed as a result of phospholipase
A2–mediated hydrolysis of the sn-2–positioned
fatty acid in phosphatidylcholine.27 Exposure of LDL to
phospholipase A2 resulted in a marked increase in
mitogenic activity. During oxidation of LDL, as much as
40% of phosphatidylcholine is degraded to lysophosphatidylcholine.
This phenomenon has been attributed to activation of a phospholipase
A2 activity associated with apolipoprotein
B.28 When added to cells, lysophosphatidylcholine is
normally converted to phosphatidylcholine by acyltransferases or
metabolized by lysophospholipases, but lysophosphatidylcholine has also
been shown to influence cell function. It enhances the activation of
human T lymphocytes,29 stimulates
endothelial expression of leukocyte adhesion
molecules,30 and inhibits
endothelium-dependent
vasodilatation.31 Ohgushi and coworkers32
have demonstrated that the latter effect is explained by the ability of
lysophosphatidylcholine to activate protein kinase C in
endothelial cells. Moreover, they showed that
impairment of endothelium-dependent vasodilatation
by oxidized LDL is caused by a lysophosphatidylcholine-dependent
activation of protein kinase C. Interestingly, lysophosphatidylcholine
potentiates diacylglycerol-induced activation of protein kinase C
but has no effect in the absence of diacylglycerol.33 This
may explain why both oxidized LDL and lysophosphatidylcholine require
the presence of an additional mitogen to stimulate DNA synthesis.
Our observations also suggest oxysterols as possible mediators of the
growth-inhibitory effects of higher concentrations of
oxidized LDL. Oxidation of LDL by copper is associated with the
formation of several oxygenated sterols, in particular
7-ketocholesterol,
7-hydroxycholesterol, 5,6-epoxide, and
25-hydroxycholesterol.34 Several earlier
studies have demonstrated the growth-inhibitory and
toxic properties of oxysterols.35 36 37
The present
results show that under serum-free conditions 5,6-epoxy
cholesterol, cholestane-3ß,5,6ß-triol,
7-hydroxycholesterol,
7ß-hydroxycholesterol, and
7-ketocholesterol were all potent
inhibitors of SMC DNA synthesis, whereas
25-hydroxycholesterol was less potent in this respect.
The oxysterols also inhibited DNA synthesis in the presence of 1%
serum, but this effect was markedly less prominent than in
serum-free medium.
Two recent studies have demonstrated that antioxidants inhibit the development of intimal thickening after balloon injury in hypercholesterolemic rabbits,14 15 suggesting that lipid oxidation may promote the proliferative response of SMCs to tissue injury. Further evidence that lipid oxidation affects SMC growth has come from studies demonstrating that copper-oxidized LDL activates PDGF-A chain gene expression in SMCs.13 38 Oxidized LDL has also been shown to enhance the cell-surface expression of PDGF receptors on SMCs.13 This finding may also explain the present observation that oxidized LDL stimulated SMC DNA synthesis more effectively in the presence of 1% NCS than in serum-free medium.
Exposure of LDL to copper for 24 hours results in an extensive oxidative modification. Using LDL oxidized by copper, several investigators have reported inhibitory effects on cell functions.39 40 In contrast, exposure of endothelial cells and SMCs to LDL only minimally modified by oxidation has been found to induce secretion of monocyte chemotactic protein-1.6 The present results also demonstrate the presence of stimulatory activity in LDL more extensively oxidized by exposure to copper but show that this activity is counteracted by cytotoxic substances at higher concentrations and oxidation for more than 8 hours.
The present study adds further support to the hypothesis that oxidized LDL promotes SMC growth and suggests that the growth-stimulatory effect of oxidized LDL is due to the formation of peroxides and lysophosphatidylcholine during the first 48 hours of oxidation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank our colleagues Federico Calara and James Stafford of Cedars-Sinai Medical Center and Britt Elving of King Gustaf V Research Institute for enthusiasm and expert technical assistance.
Received May 4, 1995; accepted November 7, 1995.
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