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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1397-1403

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*INDOMETHACIN
*VEGETABLE OIL
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1397-1403.)
© 1995 American Heart Association, Inc.

Articles

Dietary {gamma}-Linolenic Acid Modulates Macrophage–Vascular Smooth Muscle Cell Interactions

Evidence for a Macrophage-Derived Soluble Factor That Downregulates DNA Synthesis in Smooth Muscle Cells

Yang-Yi Fan; Kenneth S. Ramos; Robert S. Chapkin

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.


*    Abstract
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 down arrowResults
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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 {gamma}-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


*    Introduction
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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–{alpha}, 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{alpha} 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.


*    Methods
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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{alpha}, 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 {alpha}–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 1Down). The fatty acid composition of the diets, as determined by gas chromatography,35 is shown in Table 2Down. 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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  		Table 1. Composition of Diets


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  		Table 2. Dietary Fatty Acid Composition

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{alpha} 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.


*    Results
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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 1Down. 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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  		Figure 1. Bar graph shows effect of dietary lipid on the ability of macrophages to modulate SMC DNA synthesis. Macrophages were isolated from mice fed four different diets (CO, PO, FC, and FP) and cocultured with naive SMCs. Cocultures were incubated in the presence of [3H]thymidine with or without (control) zymosan for 39 hours and rates of DNA synthesis were measured at the end of the incubation period. Results are expressed as mean±SEM (n=6) from two separate experiments. Values with different superscripts are significantly different (P<.05).

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 2Down, top) but was without effect on LTC4 synthesis (Fig 2Down, bottom). In contrast, preincubation of macrophages with L655,238 selectively reduced zymosan-stimulated macrophage LTC4 synthesis compared with control (Fig 2Down, 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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  		Figure 2. Bar graphs show effect of eicosanoid inhibitors on macrophage PGE2 (top) and LTC4 (bottom) synthesis. Naive macrophages were untreated (None) or preincubated with indomethacin (Indo), L655,238 (L655), or indomethacin plus L655,238 (Both) for 30 minutes, washed, and incubated for 27 hours in the presence or absence (control) of zymosan. Incubation supernatants were collected and processed for PGE2 and LTC4 measurement with an EIA. Results are expressed as mean±SEM of three replicate cultures per group. Values with different superscripts are significantly different (P<.05).

Cyclooxygenase and lipoxygenase inhibitors were used to determine whether eicosanoids generated from diet-modulated macrophages differentially influenced SMC DNA synthesis in coculture (Fig 3Down). Consistent with previous data (Fig 1Up), 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 1Up and 3Down 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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  		Figure 3. Bar graph shows combined effect of dietary lipid supplementation and eicosanoid inhibitor treatment on the ability of macrophages to modulate SMC DNA synthesis. Macrophages were isolated from mice fed four different diets (CO, PO, FC, FP) and were untreated (Control) or preincubated with indomethacin (Indo), L655,238 (L655), or indomethacin plus L655,238 (Both) for 30 minutes. Macrophages were washed and subsequently cocultured with naive SMCs and incubated in the presence of [3H]thymidine without zymosan for 39 hours. Rates of SMC DNA synthesis were measured at the end of the incubation period. Results are expressed as mean±SEM of three replicate cultures per group. Values with distinct superscripts are significantly different (P<.05).

To determine the effect of dietary lipid composition on eicosanoid synthesis, three major cyclooxygenase-derived eicosanoids (PGE1, PGE2, and 6-keto-PGF1{alpha}) 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 4Down). The level of PGE2 synthesis was significantly (P<.05) lower in PO and FC cocultures relative to CO and FP cocultures (Fig 5Down). PO cocultures had the highest level of 6-keto-PGF1{alpha} among the four dietary groups (Fig 6Down), but the difference was minimal compared with the CO group.



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  		Figure 4. Bar graph shows effect of dietary lipid on macrophage-SMC PGE1 synthesis. Macrophages were isolated from mice fed four different diets (CO, PO, FC, and FP) and were subsequently cocultured with naive SMCs and incubated in the presence of [3H]thymidine without zymosan for 39 hours. Incubation supernatants were collected at the end of the incubation period and processed for PGE1 analysis. Results are expressed as mean±SEM of three replicate cultures per group. Values with different superscripts are significantly different (P<.05).



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  		Figure 5. Bar graph shows effect of dietary lipid on macrophage-SMC PGE2 synthesis. Macrophages were isolated from mice fed four different diets (CO, PO, FC, FP) and were subsequently cocultured with naive SMCs and incubated in the presence of [3H]thymidine without zymosan for 39 hours. Incubation supernatants were collected at the end of the incubation period and processed for PGE2 analysis. Results are expressed as mean±SEM of three replicate cultures per group. Values with different superscripts are significantly different (P<.05).



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  		Figure 6. Bar graph shows effect of dietary lipid on macrophage-SMC 6-keto-PGF1{alpha} synthesis. Macrophages were isolated from mice fed four different diets (CO, PO, FC, FP) and were subsequently cocultured with naive SMCs and incubated in the presence of [3H]thymidine without zymosan for 39 hours. Incubation supernatants were collected at the end of the incubation period and processed for 6-keto-PGF1{alpha} analysis. Results are expressed as mean±SEM of three replicate cultures per group. Values with different superscripts are significantly different (P<.05).

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 7Down. As is also seen in Fig 3Up, 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 8Down).



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  		Figure 7. Bar graph shows dose-dependent effect of dietary GLA on the ability of macrophages to modulate SMC proliferation. Macrophages were isolated from mice fed five different diets containing increasing concentrations of GLA (the CO, PO, CT21, CT12, and TG diets contained <0.1%, 9.6%, 9.1%, 17.5%, and 26.9% GLA, respectively) and were treated with indomethacin (50 µmol/L) (Indo) or vehicle (Control) for 30 minutes. Macrophages were then cocultured with naive SMCs and the cocultures were incubated for an additional 96 hours. Fresh medium was applied every 48 hours. SMCs were trypsinized and counted with a hemacytometer at the end of the incubation period. Results are expressed as mean±SEM of three replicate cultures per group. Values with different superscripts are significantly different (P<.05).



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  		Figure 8. Bar graph shows effect of exogenous PGE1 on macrophage-modulated SMC proliferation. Macrophages were isolated from PO-fed mice and 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 (Control). Fresh medium containing PGE1 was added to the cultures every 48 hours. SMCs were trypsinized and counted with a hemacytometer at the end of the 96-hour incubation period. Results are expressed as mean±SEM of three replicate cultures per group. Values with different superscripts are significantly different (P<.05).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 1Up). 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 1Up). 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 2Up, bottom) but not that of PGE2 (Fig 2Up, 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 1Up). 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 3Up). 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 4Up). It is noteworthy that although PGE1, PGE2, and 6-keto-PGF1{alpha} 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 4Up) may elicit the antiproliferative response. We also demonstrated that the antiproliferative effect of GLA-enriched diets was dose dependent (Fig 7Up). The addition of PGE1 to SMCs reversed the indomethacin blockade of the ability of GLA-enriched macrophages to downregulate SMC proliferation (Fig 8Up). 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 3Up). 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–{alpha} and interleukin-1ß mRNAs in macrophages after lipopolysaccharide stimulation.8 This is noteworthy because tumor necrosis factor–{alpha} 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.


*    Selected Abbreviations and Acronyms
 
CO = corn oil
CT12 = corn oil/{gamma}-linolenic acid enriched triglyceride mixture (1:2, wt/wt)
CT21 = corn oil/{gamma}-linolenic acid enriched triglyceride mixture (2:1, wt/wt)
DGLA = dihomo-{gamma}-linolenic acid
EIA = enzyme immunoassay
FC = fish oil/CO mixture (9:1, wt/wt)
FP = fish oil/PO mixture (1:3, wt/wt)
GLA = {gamma}-linolenic acid
LTC4 = leukotriene C4
PGE1 = prostaglandin E1
PGE2 = prostaglandin E2
6-keto-PGF1{alpha} = 6-keto-prostaglandin F1{alpha}
PO = primrose oil
PUFAs = polyunsaturated fatty acids
SMCs = smooth muscle cells
TG = {gamma}-linolenic acid–enriched triglyceride


*    Acknowledgments
 
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 {gamma}-linolenic acid–enriched triglyceride by Ron McKinnon, Callanish Ltd.

Received February 25, 1995; accepted June 30, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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