© 1995 American Heart Association, Inc.
Articles |
Dietary 
-Linolenic Acid Modulates Macrophage–Vascular Smooth Muscle Cell Interactions
Evidence for a Macrophage-Derived Soluble Factor That Downregulates DNA Synthesis in Smooth Muscle Cells
From the Faculty of Nutrition and Molecular and Cell Biology Group (Y.-Y.F., R.S.C.) and the Department of Veterinary Physiology and Pharmacology (K.S.R.), Texas A&M University, College Station.
Correspondence to Dr Robert S. Chapkin, Faculty of Nutrition, 442 Kleberg Center, Texas A&M University, College Station, TX 77843-2471. E-mail chapkin@zeus.tamu.edu.
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Abstract |
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Abstract Macrophages and smooth muscle cells (SMCs) are two of the major reactive cell types in atherosclerosis, a disease characterized by uncontrolled proliferation of SMCs. The present study was designed to determine how dietary oils containing
-linolenic acid
(GLA) (primrose oil [PO]) and long-chain n-3 fatty acids (fish oil)
influence the ability of macrophages to modulate SMC DNA
synthesis in vitro. Mice were fed one of four diets containing 10%
(wt/wt) corn oil (CO), PO, fish oil–CO mix (FC; 9:1, wt/wt), or fish
oil–PO mix (FP; 1:3, wt/wt) for 2 weeks. Resident peritoneal
macrophages were isolated from these mice and seeded on a
semipermeable membrane with a 30-kDa cutoff. Macrophages were
preincubated with or without 50 µmol/L indomethacin
(a cyclooxygenase inhibitor) or 50
µmol/L L655,238 (a 5-lipoxygenase
inhibitor) for 30 minutes and subsequently cocultured with
naive murine aortic SMCs grown on culture dishes. DNA synthesis in SMCs
and prostaglandin formation in coculture supernatants were
measured at the end of a 39-hour incubation period. SMC DNA synthesis
was inhibited by 28% and 60% in PO and FP diets containing 10.1% and
8.2% GLA, respectively, relative to the control CO diet containing no
GLA or long-chain n-3 fatty acid. A fourfold increase in the levels of
PGE1, a potent antiproliferative eicosanoid derived
from GLA, was observed in the PO and FP groups relative to the control
CO group. Although PGE1 levels were not different between
the CO and FC dietary groups, 15% inhibition of SMC DNA synthesis,
relative to that in mice fed the control CO diet, was observed in mice
fed the FC diet containing 13.3% 20:5n-3 and 7.6% 22:6n-3 fatty
acids. Macrophage inhibition of SMC DNA synthesis and
proliferation in mice consuming GLA-enriched diets was blocked by
indomethacin but not by L655,238. Addition of exogenous
PGE1 (100 nmol/L) reversed the effect of
indomethacin. In experiments in which mice were fed
increasing levels of GLA-containing triglycerides, the
ability of macrophages to downregulate SMC proliferation was
modulated in a dose-dependent fashion. These data indicate that
macrophages isolated from animals consuming diets supplemented
with dietary oils containing GLA reduce SMC DNA synthesis and
proliferation in a cyclooxygenase-dependent manner
and therefore may favorably modulate the atherogenic process.
Key Words: prostaglandin E1 • primrose oil • fish oil • atherosclerosis • cyclooxygenase
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Introduction |
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Alterations in the content of fatty acids in the diet can modulate membrane-bound receptors, enzyme activities, and eicosanoid production.1 2 3 4 One important approach to the dietary modification of the eicosanoid system has been the manipulation of dietary GLA (18:3n-6) and fish oil–derived long-chain n-3 PUFAs. These fatty acids are thought to play a protective role against diseases such as atherosclerosis.5 6 7 8 9 10 For example, dietary supplementation with GLA-enriched oils increased the plasma HDL cholesterol concentrations in rabbits11 and had an antihypertensive effect in rats.12 Also, dietary supplementation with n-3 PUFAs has been shown to prevent the development of atherosclerotic lesions in mice8 and the recurrence of stenosis after coronary angioplasty in humans,13 possibly by inhibition of release of atherogenic mitogens.14 However, the underlying mechanism or mechanisms responsible for the potential beneficial effects of these dietary lipids have not been fully elucidated.
Atherosclerosis, the principal cause of heart attack, stroke, and gangrene of the extremities, is responsible for 50% of deaths in the US, Europe, and Japan.15 The uncontrolled proliferation of SMCs, the major reactive cell type in atherosclerosis,16 is considered a key event in the development of atherosclerotic lesions.15 Although an abundance of information has accumulated regarding mediators that stimulate vascular SMC proliferation,15 17 less is known about inhibitory factors involved in the regulation of SMC growth.
Macrophages are present at all stages of
atherosclerosis and have been recognized as the
principal inflammatory mediators in the atheromatous
plaque microenvironment.15 Macrophages can secrete
several growth-regulatory molecules, including eicosanoids,
interleukin-1, nitric oxide, tumor necrosis factor–
, and
transforming growth factor–ß. Eicosanoids, like other mediators,
possess the ability to regulate arterial SMC
phenotype and proliferative capacity.16
PGE1, PGE2, and
6-keto-PGF1 inhibit SMC proliferation.18 19 20 21
In our own studies, we have shown that mouse peritoneal
macrophages can elongate dietary GLA to DGLA (20:3n-6) and,
upon stimulation, convert DGLA to PGE1.22 23 24
It is therefore possible that manipulation of macrophage
PGE1 synthesis by dietary GLA can influence the regulation
of SMC proliferation.
Although the mouse is generally resistant to atherosclerosis,25 the recent development of transgenic mouse models provides a unique opportunity for studying the complex dietary and genetic interactions underlying atherosclerosis.26 Techniques for gene manipulation in vivo are more advanced in the mouse than in any other mammal, and therefore this model system is being aggressively used by researchers in the field of atherosclerosis.27 28 Because alteration of SMC proliferation is a pivotal factor in the process of atherogenesis,15 29 an evaluation of the effect of diet on the ability of macrophages to influence SMC DNA synthesis in a murine model system is warranted.
We have recently developed a coculture system for studying macrophage-SMC interactions in which SMCs can be cocultured with macrophages. In this system the two cell types are separated by a semipermeable membrane with a 30-kDa cutoff.30 Macrophage-derived soluble factors such as eicosanoids are therefore allowed to pass through the membrane and influence SMC behavior while direct cell-to-cell contact is precluded. In this report we used this system to determine how dietary oils containing GLA and n-3 PUFAs influence the ability of macrophages to modulate SMC DNA synthesis and proliferation in vitro.
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Methods |
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Materials
Medium 199 was purchased from Gibco BRL. Heat-inactivated fetal bovine serum was obtained from Intergen. Collagenase was from Worthington. Trypsin EDTA solution, antibiotic/antimycotic solution, glutamine, zymosan, and indomethacin were obtained from Sigma Chemical Co. L655,238 was purchased from Biomol Research Laboratories. Prostaglandin standards, 6-keto-PGF1
,
PGE2, and LTC4 EIA kits were obtained
from Cayman Chemicals. PGE2 EIA kits were purchased from
PerSeptive Biosystems. [3H]Thymidine was from ICN
Radiochemicals. Reverse-phase extraction columns were obtained from
J.T. Baker. Fatty acid methyl ester standards were from NuChek Prep.
Nunc tissue culture inserts (Nunc catalog No. 161395) with a
30-kDa–cutoff semipermeable membrane and all Optima grade solvents
were obtained from Fisher Scientific. C57BL/6 female mice were from
Charles River (Frederick Research Facility). CO and PO were generously
provided by Traco Labs. Vacuum-deodorized Menhaden fish oil was
provided by the National Institutes of Health Fish Oil Test Material
Program, Southeast Fisheries Center. TG was provided by Callanish
Ltd.
SMC Culture
SMCs were isolated from thoracic aortas of chow-fed
pathogen-free C57BL/6 female mice by a series of enzymatic digestions
with collagenase and trypsin as previously
described.31 32 Endothelium and adventitia
were removed before isolation of SMCs. The identity of the SMC
population was confirmed by immunofluorescent labeling of
mouse –smooth muscle actin.33 Cells were grown in
Medium 199 supplemented with 10% fetal bovine serum, 2 mmol/L
glutamine, 10 000 units/mL penicillin, 10 mg/mL streptomycin, and 25
µg/mL amphotericin B. Confluent cultures were trypsinized and seeded
in 35-mm culture dishes at a density of 1x104 cells/dish
and maintained in medium containing 5% serum for the remaining test
period.
Animals and Diets
All experimental procedures in which laboratory animals were
used were approved by the Institutional Animal Care Committee of Texas
A&M University. Pathogen-free female C57BL/6 mice, weighing 12 to 14 g,
were fed one of four purified diets for 2 weeks. Diets were adequate in
all nutrients34 and varied only in the oil composition:
CO, PO, FC, or FP at 10% of the diet by weight (Table 1
). The fatty acid composition of the diets, as
determined by gas chromatography,35 is
shown in Table 2. In follow-up studies, mice were fed
one of five purified diets for 2 weeks. Diets were adequate in all
nutrients34 and varied only in the oil composition: CO,
PO, TG, CT21, or CT12 at 10% of the diet by weight. The percentage of
GLA in the CO, PO, CT21, CT12, and TG diets was <0.1%, 9.6%, 9.1%,
17.5%, and 26.9%, respectively, as determined by gas
chromatography.35
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Macrophage Isolation and Coculture With SMCs
At the end of the 2-week feeding period, mouse peritoneal
macrophages were separated from resident cells by adherence as
previously described.36 Macrophages were pooled
(six mice per group) and plated on 25-mm culture inserts at the density
of 1x106 cells/well in 2 mL of Medium 199
supplemented with 5% fetal bovine serum, 2 mmol/L glutamine, 10 000
U/mL penicillin, 10 mg/mL streptomycin, and 25 µg/mL amphotericin B
(complete medium). After 2 hours at 37°C in 5%
CO2, nonadherent cells were removed by vigorous
rinsing with HBSS. Macrophage monolayers were incubated in
complete medium with or without unopsonized zymosan (300 mg/L) and
placed onto SMC cultures as described.30 A semipermeable
membrane with a 30-kDa cutoff separated macrophages (upper
chamber) from SMCs (lower chamber). [3H]Thymidine (0.5
µCi/mL) was added to the SMC cultures immediately before the addition
of macrophages and the cocultures were incubated for an
additional 39 hours. SMCs were harvested at the end of this period and
[3H]thymidine incorporation and protein concentration
were measured as previously described.37 38
Influence of Inhibitors on Macrophage
Eicosanoid Synthesis
Macrophages were preincubated in complete medium for 2
hours and then treated with the cyclooxygenase
inhibitor indomethacin (50 µmol/L), the
5-lipoxygenase inhibitor L655,238 (50
µmol/L), indomethacin plus L655,238 (50 µmol/L
each), or vehicle for 30 minutes. These concentrations and the duration
of treatment were defined in preliminary studies.23 After
preincubation, macrophages were washed and subsequently
incubated in complete medium with or without zymosan (300 mg/L) for 27
hours. Supernatants were collected at the end of the incubation period
and eicosanoid levels were measured by use of an EIA (Cayman
Chemicals). The PGE2 EIA kit used in this assay is highly
specific for PGE2, with less than 6.5%
cross-reactivity with PGE1 and other
prostaglandins.
Effect of Diet and Eicosanoid Inhibitors on SMC DNA
Synthesis and Proliferation
Mice were fed four different diets for 2 weeks and
macrophages were isolated at the end of the feeding period as
described above. Macrophages were seeded onto tissue culture
inserts and treated with indomethacin (50 µmol/L),
L655,238 (50 µmol/L), indomethacin plus L655,238 (50
µmol/L each), or vehicle for 30 minutes. After preincubation,
macrophages were washed and subsequently cocultured with pooled
naive SMCs (isolated from chow-fed mice) in the presence of
[3H]thymidine (0.5 µCi/mL) without zymosan for 39 hours
as described above. At the end of the incubation period, supernatants
were collected and processed for prostaglandin
analysis.23 SMCs were harvested and DNA synthesis
and protein concentration were measured as described
previously.37 38 For cell proliferation experiments,
macrophages were isolated from mice at the end of a 2-week
feeding period and preincubated in the presence or absence of
indomethacin (50 µmol/L) for 30 minutes. These
macrophages were then cocultured with naive SMCs. After a
96-hour incubation, SMCs were trypsinized and counted with a
hemacytometer. For the prostaglandin rescue experiments,
macrophages isolated from PO-fed mice were pretreated with
indomethacin (50 µmol/L) for 30 minutes, washed, and
cocultured with naive SMCs in the presence of exogenous
PGE1 (1 nmol/L, 100 nmol/L, or 10 µmol/L) or vehicle. The
cocultures were incubated for 96 hours and SMCs were subsequently
trypsinized and counted with a hemacytometer.
Measurement of Prostaglandin Synthesis
Incubation supernatants were applied to C-18 reverse-phase
extraction columns for elution of
prostaglandins.39 Prostaglandins
were separated on a reverse-phase C18 Ultrasphere ODS column (5-µm
particle size, 4.6 mm [ID]x25 cm; Beckman Instruments) by use of an
isocratic solvent mixture of acetonitrile:0.0174 mol/L
o-phosphoric acid (32.8:67.2, vol/vol). Samples were run for
30 minutes at a flow rate of 1 mL/min and continuously monitored at 196
nm.22 PGE1 and PGE2 were eluted as
separate peaks and column fractions were collected into siliconized
polypropylene tubes.23 Fractions containing
PGE1 and PGE2 were subsequently measured by an
EIA (PerSeptive Biosystems). Antibody cross-reactivity with
PGE2 and PGE1 in this PGE2 EIA kit
is 100% and 50%, respectively. Therefore, the result for
immunoreactive PGE1 was multiplied by 2 to determine
PGE1 mass. 6-keto-PGF1 in incubation
supernatants was directly assayed by use of an EIA (Cayman
Chemicals).
Statistical Analysis
Data were analyzed by two-way ANOVA with the
STATISTICAL ANALYSIS
SOFTWARE package (SAS Institute) and by Duncan's
multiple range test. A difference of P<.05 was considered
statistically significant.
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Results |
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No statistical differences in animal body weights were observed among the four dietary groups at the end of the feeding period, suggesting that different dietary oils were associated with comparable growth rates (data not shown). With the coculture experimental model, SMC [3H]thymidine incorporation was 2457±201 and 3191±108 cpm/µg protein (n=3) in the absence and presence of macrophages from chow-fed mice, respectively. The effect of dietary lipid on the ability of macrophages to modulate SMC DNA synthesis is shown in Fig 1
. When macrophages
were not stimulated with zymosan (control), PO- and FP-fed mice
exhibited a significant (P<.05) reduction in SMC DNA
synthesis compared with mice fed the CO diet (containing no GLA or n-3
PUFAs). When SMCs were cocultured with zymosan-stimulated
macrophages, PO-, FC-, and FP-fed mice had a significantly
lower (P<.05) level of DNA synthesis relative to the CO
group. Macrophages harvested from FP-fed mice had the strongest
inhibitory effect on SMC DNA synthesis, irrespective of the
degree of macrophage stimulation. Macrophages from
FC-fed mice only inhibited SMC DNA synthesis in the presence of
zymosan.
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A previous study in this laboratory indicated that 50 µmol/L of
indomethacin significantly inhibited eicosanoid
synthesis in mouse peritoneal macrophages.23
Preincubation of macrophages with this concentration of
indomethacin significantly (P<.05) reduced
PGE2 levels compared with untreated cultures (Fig 2, top) but was without effect on LTC4
synthesis (Fig 2, bottom). In contrast, preincubation of
macrophages with L655,238 selectively reduced
zymosan-stimulated macrophage LTC4 synthesis
compared with control (Fig 2, bottom), with minimal effects on
PGE2 synthesis. The combination of both
inhibitors did not influence the inhibitory
patterns of individual agents on PGE2 and LTC4
synthesis. Zymosan supplementation enhanced LTC4 but not
PGE2 synthesis compared with controls.
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Cyclooxygenase and lipoxygenase
inhibitors were used to determine whether eicosanoids
generated from diet-modulated macrophages differentially
influenced SMC DNA synthesis in coculture (Fig 3).
Consistent with previous data (Fig 1
), when macrophages
were coincubated in the absence of cyclooxygenase
and/or lipoxygenase inhibitors, SMC DNA
synthesis was inhibited (P<.05) by 28%, 15%, and 60% in
the PO, FC, and FP dietary groups, respectively, relative to the CO
group. Data shown in Figs 1 and 3 were from two separate experiments
with different cell strains. Although absolute
[3H]thymidine incorporation values varied in these two
populations, the overall profile (CO>FC>PO>FP) was similar. Thus,
the effect of dietary lipid on the ability of macrophages to
modulate SMC DNA synthesis was highly reproducible irrespective of
absolute rates of DNA synthesis. The inhibition of SMC DNA synthesis by
GLA-enriched diets was diminished by preincubation with
indomethacin but not with L655,238, and the combination
of inhibitors induced an intermediate
inhibitory effect. These data suggest that the
macrophage-derived cyclooxygenase
metabolites may, at least in part, mediate the ability of dietary lipid
to downregulate vascular SMC DNA synthesis.
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To determine the effect of dietary lipid composition on eicosanoid
synthesis, three major cyclooxygenase-derived
eicosanoids (PGE1, PGE2, and
6-keto-PGF1) in the control and
indomethacin-treated coculture systems were
measured. Dietary manipulation significantly (P<.05)
altered prostaglandin levels only in the control group.
Incubation supernatants from the PO and FP dietary groups, which
contained 10.1% and 8.2% GLA, had significantly (P<.05)
elevated PGE1 levels relative to supernatants from the CO
and FC groups (Fig 4). The level of PGE2
synthesis was significantly (P<.05) lower in PO and FC
cocultures relative to CO and FP cocultures (Fig 5). PO
cocultures had the highest level of 6-keto-PGF1 among
the four dietary groups (Fig 6), but the difference was
minimal compared with the CO group.
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When mice were fed diets containing varying concentrations of TG, the
ability of macrophages to downregulate SMC proliferation was
dose dependent, as shown in Fig 7. As is also seen in
Fig 3, addition of indomethacin abolished the
inhibitory effect of GLA-enriched macrophages on
SMCs. Exogenous PGE1 reinstated the antiproliferative
response precluded by indomethacin treatment of
GLA-enriched macrophages (Fig 8).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Dietary lipids can alter the PUFA composition of membrane phospholipids and modulate cellular eicosanoid biosynthesis.23 35 36 40 Eicosanoids have been implicated in the regulation of vascular homeostasis,41 including the regulation of arterial SMC phenotype, cellular cholesterol metabolism, and SMC proliferation.17 The mechanisms by which select dietary fatty acids can favorably modulate excessive inflammatory-fibroproliferative diseases such as atherosclerosis are poorly understood. Because macrophages are present at all stages of atherosclerosis and are considered the principal inflammatory mediators in the atheromatous plaque environment,15 it has long been suspected that products secreted from macrophages play an important role in the regulation of SMC proliferation.17 We have previously demonstrated that mouse peritoneal macrophages can elongate dietary GLA to DGLA, which is converted to PGE1 upon stimulation.22 23 24 In addition, the dietary combination of GLA and n-3 PUFAs preferentially enhances macrophage biosynthesis of PGE1 relative to PGE2.23 This is noteworthy because PGE1 has been used in the treatment of cardiovascular disease and chronic inflammation.42 43 However, PGE1 has a very short half-life in the body and can only be effectively administered intravascularly. Although several stable PGE1 analogues have been used as immunosuppressive agents,44 45 these analogues have unpredictable and sometimes adverse effects.45
The present study was designed to determine how dietary oils
containing GLA and n-3 PUFAs modulate macrophage-SMC eicosanoid
synthesis and influence the ability of macrophages to modulate
SMC DNA synthesis in vitro. The results presented here
demonstrate that dietary oils containing GLA can reduce SMC DNA
synthesis and that the inhibitory effect is not dependent
on the level of macrophage stimulation (Fig 1). As with the
GLA-enriched diets, FC-derived macrophages (containing n-3
PUFAs) also reduced SMC DNA synthesis relative to the CO control, but
the effect was dependent on the level of macrophage stimulation
(Fig 1). In a previous study23 we showed that when
macrophages are incubated in serum-free medium, the addition of
zymosan significantly elevates the synthesis of
prostaglandins. However, the addition of zymosan does not
enhance prostaglandin synthesis when macrophages
are maintained in 5% serum. Thus, if serum activates
macrophage cyclooxygenases, zymosan
stimulation of macrophages in 5% serum–containing medium
enhances the synthesis of LTC4 (Fig 2, bottom) but not that
of PGE2 (Fig 2, top). This interpretation suggests that the
antiproliferative effect of GLA and fish oil on SMC DNA synthesis may
be mediated through distinct mechanisms. Interestingly, the combination
of fish and GLA-enriched oils in the FP diet produced an additive
downregulation of SMC DNA synthesis, resulting in the lowest rates of
DNA synthesis (Fig 1). In the macrophage-SMC coculture system
used in these studies, SMCs were isolated and pooled from the same
chow-fed mice. In contrast, macrophages were isolated from mice
fed the different diets. Therefore, the regulatory effect of dietary
GLA and n-3 PUFAs on SMC DNA synthesis in this coculture model can be
attributed to the modulation of macrophage-derived soluble
mediators.
Results of eicosanoid inhibitor experiments demonstrated
that macrophages isolated from mice fed GLA-enriched diets
downregulated naive SMC DNA synthesis in a
cyclooxygenase-dependent manner (Fig 3).
Consistent with our previous observations,23
PGE1 biosynthesis in macrophage-SMC coculture was
significantly (P<.05) enhanced in the mice consuming
GLA-enriched diets (Fig 4). It is noteworthy that although
PGE1, PGE2, and
6-keto-PGF1 all possess inhibitory
properties with respect to SMC DNA synthesis, PGE1 has the
greatest biopotency.19 21 46 Therefore, it is possible
that the elevated levels of PGE1 associated with the PO and
FP cocultures compared with the CO cocultures (Fig 4) may elicit the
antiproliferative response. We also demonstrated that the
antiproliferative effect of GLA-enriched diets was dose dependent (Fig 7). The addition of PGE1 to SMCs reversed the
indomethacin blockade of the ability of GLA-enriched
macrophages to downregulate SMC proliferation (Fig 8). These
results strongly suggest that cyclooxygenase
metabolites derived from dietary GLA play an important role in the
observed macrophage-SMC interactions. In addition, although the
level of PGE1 in the FC coculture was not elevated relative
to the control CO coculture, the SMC DNA synthesis in the FC group was
decreased compared with that in the CO group (Fig 3). These data
suggest that the antiproliferative effect of dietary fish oil on SMCs
may involve the release of a macrophage-derived soluble
factor or factors other than PGE1. For example, animals fed
fish oil–containing diets have dramatically decreased expression of
tumor necrosis factor– and interleukin-1ß mRNAs in
macrophages after lipopolysaccharide
stimulation.8 This is noteworthy because tumor necrosis
factor– and interleukin-1 can induce hemorrhagic necrosis and
stimulate the proliferation of SMCs.47 48 Interestingly,
interleukin-1 has also been shown to induce PGE production in
human SMCs, an effect that can counteract the intrinsic
mitogenicity of interleukin-1 in SMCs upon short-term (2
days) but not long-term (7 to 28 days) incubation.48 Thus,
it is possible that the growth-inhibitory response of
GLA-enriched diets is secondary to increased interleukin-1
production in macrophages that in turn upregulates
prostanoid production in SMCs. In this regard, it is also
conceivable that the inhibitory potential of FP-derived
macrophages on SMCs involves the additive effect of elevated
PGE1 levels by GLA supplementation and decreased
pro-proliferative cytokines by fish oil supplementation.
Further studies are required to elucidate the mechanism or mechanisms
by which dietary PO and fish oil downregulate vascular SMC growth
programs.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This research was supported in part by grants from Scotia Pharmaceutical Ltd to Dr Chapkin, the Texas A&M Interdisciplinary Research Enhancement Program to Dr Ramos and Dr Chapkin, and National Institutes of Health grant DK-41693 to Dr Chapkin. Y.-Y. Fan is a recipient of the 1994 Nabisco Foods Group Predoctoral Fellowship sponsored by the American Institute of Nutrition. We also gratefully acknowledge the generous donation of corn and primrose oils by Sid Tracy, Traco Labs, and
-linolenic acid–enriched
triglyceride by Ron McKinnon, Callanish Ltd. Received February 25, 1995; accepted June 30, 1995.
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