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-Linolenic Acid Enhances Mouse Macrophage-Derived Prostaglandin E1 Which Inhibits Vascular Smooth Muscle Cell Proliferation1,2
Faculty of Nutrition and Molecular and Cell Biology Group, Texas A&M University, College Station, TX 77843-2471 and * Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
We previously demonstrated that macrophages isolated from mice fed 
-linolenic acid (GLA)-enriched diets reduce vascular smooth muscle cell (SMC) proliferation in a cyclooxygenase-dependent fashion and may therefore favorably modulate the atherogenic process. The present study was conducted to elucidate the mechanism(s) by which dietary GLA influences the ability of macrophages to modulate SMC growth programs. Resident peritoneal macrophages were isolated from C57BL/6 female mice fed diets containing variable GLA compositions at 10% (wt/wt), treated with various antibodies and co-cultured with cycling naive vascular SMC isolated from nonpurified diet-fed mice. Smooth muscle cell proliferation and intracellular cAMP levels were measured after co-culture. In parallel experiments, cycling naive vascular SMC isolated from nonpurified diet-fed mice were dosed with exogenous prostaglandin E1 (PGE1 ) for various periods and challenged with cycloheximide for 4 h (8-12 h after PGE1 addition), and intracellular cAMP levels were measured at various time points. Macrophages isolated from mice fed GLA-enriched dietary oils significantly reduced SMC proliferation in co-culture compared with controls (macrophages from mice fed a corn oil diet containing no GLA). Anti-PGE1 antiserum treatment (1:50 or 1:100) blocked the ability of GLA-enriched macrophages to down-regulate SMC proliferation, a response reversed by exogenous PGE1 treatment. Macrophages isolated from mice fed GLA-enriched dietary oils elevated SMC intracellular cAMP levels in a biphasic fashion. In addition, exogenous PGE1 (1 nmol/L to 10 µmol/L) exerted a similar biphasic cAMP response in SMC, and the second phase of cAMP elevation was antagonized by cycloheximide. In conclusion, dietary GLA enhances mouse macrophage-derived prostaglandin E1 , which inhibits vascular SMC proliferation.
-linolenic acid ·
mice ·
proliferation ·
prostaglandin E1
INTRODUCTION
Atherosclerosis is an insidious disease that accounts for most of the morbidity and mortality seen in humans with coronary heart disease and stroke. Numerous genetic and environmental factors have been suggested to influence the development of atherosclerosis (Badimon et al. 1993
, Hegele 1992, Ou and Ramos 1992, Ramos et al. 1993, Ross 1993). Several animal models have been used to study the molecular and cellular events that mediate pathologic progression of this disease. Although mice are naturally resistant to atherosclerosis, techniques for gene manipulation are more advanced in this model system than in any other mammal. Therefore, over the last decade, the mouse model has been aggressively utilized for the elucidation of the common and complex polygenic mechanisms of cardiovascular diseases (Lusis 1993, Paigen et al. 1994, Rubin and Smith 1994).
Dietary fatty acids can modulate the fatty acid composition of membrane phospholipids and consequently alter the profile of eicosanoid biosynthesis in macrophages (Chapkin et al. 1988a, Fan and Chapkin 1992, Willis 1981). Many human and murine tissues have an active long-chain polyunsaturated fatty acid elongase but limited 
6- and 5-desaturase activities (Chapkin et al. 1988b, Horrobin 1990) (Fig. 1). Therefore, the rate of -linolenic acid [GLA,4 18:3(n-6)] formation from linoleic acid [18:2(n-6)] may be inadequate to provide sufficient amounts of GLA and its metabolites (Horrobin 1990). Studies have shown that the ingestion of GLA results in the accumulation of dihomo--linolenic acid [DGLA, 20:3(n-6)], with little or no change in arachidonic acid [AA, 20:4(n-6)] levels (Barre and Holub 1992, Chapkin et al. 1988a, Chilton-Lopez et al. 1996, Fan and Chapkin 1992, Ziboh and Fletcher 1992). We have previously demonstrated that diets supplemented with GLA-enriched oils enhance mouse peritoneal macrophage DGLA levels in membrane phospholipids and, upon stimulation, enhance the secretion of prostaglandin E1 (PGE1 ) (Fan and Chapkin 1992 and 1993). These effects are significant because PGE1 exerts several biological effects of importance in atherogenesis, such as the inhibition of smooth muscle cell (SMC) proliferation, platelet aggregation, superoxide anion generation, and activation of the fibrinolytic system (Fan et al. 1996b, Horrobin 1988, Nilsson and Olsson 1984, Owen 1986, Simmet and Peskar 1988).
-Linolenic acid (GLA) metabolic flow scheme. Because -5 desaturase activity is rate-limiting, dietary -linolenic acid (GLA) elevates macrophage dihomo--linolenic acid (DGLA) with little or no change in arachidonic acid (AA) levels. DGLA is a precursor to one-series prostaglandins (PGE1 ).
Vascular SMC proliferation is one of the key events implicated in the pathogenesis of atherosclerotic vessel disease (Badimon et al. 1993). Macrophages secrete a number of growth regulatory molecules capable of influencing SMC proliferation and have been recognized as the principal inflammatory mediator in the atheromatous plaque micro-environment (Ross 1993). Therefore interactions between these two cell types are thought to be critical in the initiation and progression of atherosclerotic lesions (Ross 1993). Previously, we demonstrated that when SMC isolated from mice fed a nonpurified diet are co-cultured with macrophages isolated from mice consuming GLA-enriched dietary oils, PGE1 but not PGE2 or 6-keto-PGF1
levels in incubation supernatants are significantly increased, and SMC proliferation is significantly decreased (Fan et al. 1995b). Because dietary GLA has no direct effect on vascular SMC proliferation, macrophage-derived prostaglandins were implicated in the growth inhibitory response (Fan et al. 1995b). However, definitive identification of PGE1 as a primary factor modulated by GLA-enriched dietary oils awaits further evidence.
It has been shown that the biological effects of PGE1 in various cells are mediated by activation of adenylate cyclases and subsequent elevation of intracellular cAMP levels (Owen 1986). The elevation of cAMP activates cAMP-dependent protein kinases (PKA), which can phosphorylate several transcription factors and thereby modulate cell growth and differentiation (Beebe 1994, Cox et al. 1990, Taylor 1990). We, and others, have shown that the addition of exogenous PGE1 to SMC elevates intracellular cAMP levels (Fan et al. 1996b, Owen 1986), which may contribute to the down-regulation of SMC proliferation (Loesberg et al. 1985, Nilsson and Olsson 1984). However, it is not known whether the enhancement of macrophage-derived PGE1 following GLA supplementation influences SMC intracellular cAMP levels. Therefore we conducted the present study to define the mechanism(s) by which dietary GLA down-regulates macrophage-dependent vascular SMC growth programs.
MATERIALS AND METHODS
and 1993). The SMC phenotype was confirmed by Northern-blot analysis and immunofluorescent labeling of mouse 
-smooth muscle specific actin (Lundberg et al. 1995). Cells were grown in Medium 199 supplemented with 10% FBS, 2 mmol/L glutamine, 1 × 107 U/L penicillin, 10 g/L streptomycin and 25 mg/L amphotericin B. Confluent cultures were trypsinized and seeded in 35-mm culture dishes at a density of 1 × 104 cells/dish and maintained in medium containing 5% serum for the remaining test period.
Animals and diets.
All experimental procedures using laboratory animals were approved by the Institutional Animal Care Committee of Texas A&M University. Three separate dietary studies were conducted. For each dietary study, pathogen-free female C57BL/6 mice, weighing 12-14 g, were given free access to one of the different purified diets for 2 wk. Diets were adequate in all nutrients (AIN 1977) and varied only in the oil composition at 10% of the diet by weight (Table 1). Powdered diets were mixed at the beginning of each study and distributed into plastic Zip-lockTM bags, flushed with nitrogen and stored at 
20°C. Mice were given free access to fresh diet on a daily basis. Each diet was supplemented with t-butylhydroquinone to prevent oxidative breakdown (Fritsche and Johnston 1988). In addition, diets were supplemented with vitamin E (50 mg -tocopherol/kg) to protect against in vivo peroxidation (Fritsche and Johnston 1988).
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Table 1. Diet composition |
Table 2.
Dietary fatty acid composition1
), is shown in Table 2.
). Macrophages were plated on 25-mm culture inserts at the density of 1 × 106 cells/well in 2 mL of Medium 199 supplemented with 5% FBS, 2 mmol/L glutamine, 1 × 107 U/L penicillin, 10 g/L streptomycin and 25 mg/L amphotericin B (complete medium). After 2 h at 37°C in 5% CO2 , non-adherent cells were removed by vigorous rinsing with Hank's balanced salt solution. Adherent cells were immediately used for the following co-culture studies.
-linolenic-enriched triglyceride-fed mice. Macrophages (1 × 106 cells/dish, upper chamber) were isolated from corn oil (CO) and GLA-enriched triglyceride (GLA-TG)-fed mice, treated with anti-PGE1 antiserum (1:50, 1:100, 1:500, 1:5000 dilution) or vehicle and subsequently co-cultured with naive cycling SMC (1 × 104 cells/dish, lower chamber) isolated from nonpurified diet-fed mice. Anti-PGE1 antiserum was added to macrophage cultures every 24 h. The SMC were trypsinized and counted using a hemacytometer at the end of the 96-h incubation period. Results are expressed as the percentage of cell number in GLA-TG fed mice relative to the vehicle-treated CO-fed group (mean ± SEM, n = 6). The SMC number in the control CO group was 73,800 ± 6300 (n = 6). No significant effect of anti-PGE1 was found in the CO group; therefore, only data from GLA-TG-fed mice are shown. Smooth muscle cell proliferation from the vehicle-treated CO group is shown for comparison to the TG groups. Values with different letter superscripts are significantly different (P < 0.05).
Antibody treatment. Macrophages isolated from mice fed either CO or GLA-TG were incubated in complete medium (upper chamber, 25-mm culture inserts), treated with various concentrations of anti-PGE1 antiserum (1:50, 1:100, 1:500, 1:5000 dilution) or vehicle, subsequently co-cultured with cycling naive SMC isolated from nonpurified diet-fed mice (lower chamber, 35-mm culture dishes), and incubated for 96 h as previously described (Fan et al. 1995a
). Anti-PGE1 antiserum was added to macrophage cultures (upper chamber) every 24 h and, in selected cultures, exogenous PGE1 (100 nmol/L, 1 and 10 µmol/L) or vehicle was added to SMC cultures (lower chamber) every 48 h to antagonize the effect of anti-PGE1 antiserum. Viability of SMC was monitored daily by trypan blue exclusion, and the medium was replaced every other day. The SMC were trypsinized and counted using a hemacytometer at the end of the 96-h incubation period. In a separate series of experiments, macrophages isolated from mice fed either CO or GLA-mix were incubated in complete medium and treated with anti-PGE1 antiserum (1:50, 1:100 dilution), denatured anti-PGE1 antiserum (1:50, 1:100 dilution), anti-PGD2 antiserum (1:50, 1:100 dilution), anti-PDGF antiserum (2.5 mg/L) or vehicle immediately prior to co-culture with cycling naive SMC isolated from nonpurified diet-fed mice. Denatured anti-PGE1 antiserum was obtained by heating anti-PGE1 antiserum in boiling water for 10 min immediately before use. The co-cultures were incubated for 96 h, and fresh antibody was added daily. The SMC were trypsinized and counted using a hemacytometer at the end of the 96-h incubation period.
) and measured by enzyme immunoassay (EIA) according to the kit instructions (catalogue no. 90006, Assay Designs, Ann Arbor, MI).
). Smooth muscle cell intracellular cAMP levels were measured by EIA as described above.
Statistical analysis.
Data were analyzed by one-way ANOVA using SAS (Cary, NC) software and Duncan's multiple range test (Ott 1988), except where noted in the text. A difference of P < 0.05 was considered significant.
RESULTS
-linolenic-enriched triglyceride (GLA-TG)-fed mice were treated with various concentrations of anti-PGE1 antiserum (1:5000, 1:500, 1:100) or vehicle, and co-cultured with naive cycling SMC (1 × 104 cells/dish, lower chamber) in the presence of exogenous PGE1 (100 nmol/L, 1 and 10 µmol/L) or vehicle. Medium containing PGE1 was added to the SMC cultures every 48 h. The SMC were trypsinized and counted using a hemacytometer at the end of the 96-h incubation period. Results are expressed as means ± SEM, n = 6. Values with different letter superscripts are significantly different (P < 0.05). Only results from the CO group in the absence of anti-PGE1 antiserum treatment are presented (n = 6). Similar profiles were seen when macrophages were treated with anti-PGE1 antiserum (1:5000, 1:500, 1:100).
Effect of anti-PGE1 antiserum and exogenous PGE1 on macrophage-smooth muscle cell interaction. The effect of anti-PGE1 antiserum on macrophage modulation of SMC proliferation is shown in Figure 2. In the absence of anti-PGE1 antiserum, macrophages isolated from GLA-TG-fed mice and immediately co-cultured with naive SMC from nonpurified diet-fed mice for 96 h had significantly (P < 0.05) reduced SMC proliferation relative to the vehicle-treated, CO-fed group containing primarily linoleic acid and no GLA (Student's t-test comparison). The addition of anti-PGE1 antiserum reversed the growth inhibitory effect observed in GLA-TG co-cultures in a concentration-dependent manner. Addition of anti-PGE1 antiserum did not significantly (P > 0.05) influence SMC proliferation in the CO-fed group (data not shown). In contrast, exogenous PGE1 reduced SMC proliferation in both dietary groups. Figure 3 shows the effect of exogenous PGE1 on SMC proliferation in the CO-fed group in the absence of anti-PGE1 antiserum. Similar concentration-dependent profiles were observed in the presence of anti-PGE1 antiserum (1:5000, 1:500, 1:100) (data not shown). Specificity of antibody effects on macrophage-mediated smooth muscle cell proliferation. The effect of different antibodies on SMC proliferation in co-cultures containing macrophages isolated from GLA-mix-fed mice is shown in Figure 4. No significant difference in SMC proliferation in response to various antibodies in the CO dietary group was observed (data not shown). Relative to the vehicle-treated, CO-fed group, the inhibitory effect of GLA-enriched macrophages on SMC proliferation was completely blocked only when macrophages were treated with anti-PGE1 antiserum (1:50).
-linolenic acid (GLA) free fatty acid-safflower oil ethyl ester mixture (35:65, wt/wt) (GLA-mix)-fed mice, treated with various concentrations of antibody and subsequently co-cultured with naive cycling SMC (1 × 104 cells/dish, lower chamber) isolated from nonpurified diet fed mice for an additional 96 h. Antibodies (anti-PGE1 , anti-PGD2 and anti-PDGF ) or vehicle (control) was added to macrophage cultures every 24 h, and fresh medium was applied every 48 h. The SMC were trypsinized and counted using a hemacytometer at the end of the 96-h incubation period. Results are expressed as means ± SEM, n = 6. Values with different letter superscripts are significantly different (P < 0.05). No significant differences were observed in the CO groups; therefore, only results from the GLA-mix-fed mice are presented. The SMC proliferation from the vehicle-treated CO group is shown as a comparison.
Effect of
-linolenic acid-enriched macrophages on smooth muscle cell intracellular cAMP levels.
Figure 5 shows the effect of distinct dietary lipids on macrophage-dependent modulation of SMC (from nonpurified diet-fed mice) intracellular cAMP levels. A similar profile was observed in the GLA-enriched dietary groups (PO, BO and FP); therefore, only data from the mice fed PO and CO are presented. Compared with results for the CO-fed group, a similar biphasic elevation in cAMP levels was observed in mice fed GLA-containing diets (PO, BO and FP). Despite the varying GLA composition of the PO, BO and FP diets (Table 2), the cAMP profiles in SMC from mice fed the GLA-containing diets were similar, suggesting that the bioavailability of GLA in the different dietary oils was comparable (Fan et al. 1996a) with respect to their ability to modulate SMC intracellular cAMP levels. The SMC intracellular cAMP levels were rapidly elevated (by 5 min) after co-culture with macrophages. The increase reached a peak by 20 min, gradually decreasing to basal levels by 1 h. A second phase of cAMP production was observed 16 h after co-culture and maintained until the termination of the experiment at 40 h.
Effect of cycloheximide treatment on smooth muscle cell intracellular cAMP levels. The ability of cycloheximide to block the PGE1 -induced elevation of SMC (from nonpurified diet-fed mice) intracellular cAMP levels is shown in Figure 6. No evidence of injury was observed as determined by lactate dehydrogenase leakage into the medium (data not shown). Relative to control cells (no PGE1 added), addition of PGE1 immediately induced an significant (P < 0.05) increase of intracellular cAMP, peaking at 1 h and gradually decreasing to basal levels by 2 h. In the absence of cycloheximide, a significant (P < 0.05) secondary increase in intracellular cAMP was evident at 16 h and maintained until 40 h.
cAMP) in treatment groups relative to the control group (no PGE1 and cycloheximide added) (mean ± SEM, n = 3). The PGE1 -dependent biphasic induction of cAMP was dose-dependent (data not shown). Bottom: An amplified scale for the 2-40 h period is shown. Data were analyzed by one-way ANOVA and Duncan's multiple range test with respect to the four treatment groups at each time point. Only the data from the 10 µmol/L PGE1 and vehicle treatment groups are presented. An asterisk indicates statistical significance (P < 0.05).
DISCUSSION
). Macrophages and SMC are considered two of the major reactive cell types involved in this disease process. Our previous studies have shown that dietary GLA can modulate soluble factors secreted by macrophages that influence the proliferation of SMC in co-culture (Fan et al. 1995b and 1996a). A likely candidate responsible for the down-regulation of SMC proliferation by GLA-enriched macrophages is PGE1 , an eicosanoid derived from DGLA that exerts anti-proliferative effects in a variety of cell types, including SMC (Horrobin 1988, Simmet and Peskar 1988). In view of the prominent role of SMC proliferation in atherogenesis (Badimon et al. 1993, Ross 1993), dietary GLA manipulation may favorably modulate the atherogenic process. Therefore, the current study was conducted to elucidate the mechanism(s) by which dietary GLA influences the interactions of macrophages and SMC.
, 1995b and 1996a), SMC proliferation was significantly (P < 0.05) reduced when SMC from nonpurified diet-fed mice were co-cultured with macrophages from mice fed GLA compared with control CO, which contains no GLA (Fig. 2 and 4). These data demonstrate that dietary GLA-enriched oils increase the macrophage-dependent down-regulation of SMC proliferation, irrespective of the form of GLA supplemented (i.e., natural plant oil, enriched triglyceride or free fatty acid). Because we previously established that macrophage-derived cyclooxygenase metabolites play an important role in the down-regulation of SMC proliferation (Fan et al. 1995b), we were particularly interested in the identifying the specific prostaglandins critical to this response using a panel of specific antibodies.
). Second, exogenous PGE1 reduced SMC proliferation in CO groups to a level comparable with that of the GLA-TG group in the absence of exogenous PGE1 (Fig. 2 and 3). Third, for the GLA-mix group, except for the anti-PGE1 antiserum treatment, no significant blocking effect was observed when macrophages were treated with denatured anti-PGE1 antiserum (1:50, 1:100 dilution), anti-PGD2 antiserum (1:50, 1:100 dilution), anti-PDGF antiserum (2.5 µg/mL) or vehicle, suggesting that the anti-PGE1 effect on macrophage-dependent modulation of SMC proliferation is specific (Fig. 4). These data indicate that, with regard to the different levels of prostaglandins and growth factors tested, PGE1 is a primary, soluble mediator responsible for the anti-proliferative effect of dietary GLA.
, Nilsson and Olsson 1984, Owen 1986). However, the extent to which macrophages from the different dietary regimens influence SMC intracellular cAMP levels has not been investigated. Therefore, the effect of GLA supplementation (PO, BO and FP diets) relative to CO (control, containing no GLA) on cAMP levels was examined. These studies were designed to define the mechanism by which dietary GLA down-regulates macrophage-dependent vascular SMC growth programs. As shown in Figure 5, immediately following co-culture, macrophages isolated from PO-fed mice had significantly (P < 0.05) greater SMC (isolated from nonpurified diet-fed mice) intracellular cAMP levels relative to the CO group. Cyclic AMP levels increased in a biphasic manner, peaking at 20 min and subsequently decreasing to basal levels by 1 h. Following a lag phase of approximately 16 h, cAMP levels increased and remained elevated for the duration of the experiment. We previously demonstrated that peritoneal macrophages isolated from mice fed GLA-enriched dietary oils, such as PO, BO and FP, have comparable PGE1 levels in culture supernatants irrespective of the different GLA levels in those oils (Fan and Chapkin 1992, Fan et al. 1995b). Because PGE1 transiently increases SMC intracellular cAMP levels (Loesberg et al. 1985, Nilsson and Olsson 1984, Owen 1986), the comparable increase of cAMP in PO, BO and FP groups is likely the result of PGE1 secretion from GLA-enriched macrophages. It is unlikely that the reduction in cAMP levels over time was due to the degradation of PGE1 , because we have shown that macrophages can secrete PGE1 within 1 h after stimulation (Fan and Chapkin 1992) and that the levels of PGE1 in incubation supernatants remain elevated for at least 39 h (Fan et al. 1995b). It is possible that the refractory phase of the cAMP response results from receptor desensitization, because during continuous exposure to agonists i.e., PGE1 , a number of G-protein-coupled receptors undergo regulatory processing that results in diminution of signal transduction (Garrity et al. 1983, Liggett and Lefkowitz 1994, Thierauch et al. 1994). Further studies utilizing specific inhibitors of adenylate cyclase, phosphodiesterase and PKA will help to define the molecular mechanisms contributing to the cAMP response.
). The induction of newly synthesized proteins may feedback in an autocrine loop to re-activate adenylate cyclases or inhibit phosphodiesterases or induce a new signal that in turn influences cAMP levels (Fig. 5). To test this hypothesis, SMC intracellular cAMP levels were measured following treatment with exogenous PGE1 with or without cycloheximide (an inhibitor of protein synthesis). The effect of cycloheximide on the initial peak was not examined, because it is unlikely that protein synthesis is required for an immediate response. Consistent with the cAMP data generated in co-culture using macrophages isolated from GLA-fed mice (Fig. 5), addition of exogenous PGE1 resulted in a dose-dependent biphasic elevation of cAMP levels in SMC (Fig. 6), although the first peak was much higher than the second. It is possible that G-protein-coupled prostaglandin receptors were activated by exogenous PGE1 , leading to pronounced elevation of cAMP levels (Liggett and Lefkowitz 1994, Nilsson and Olsson 1984). In contrast, the second phase of cAMP elevation may require de novo synthesis of new proteins (e.g., receptors, adenylate cyclases) and thus represents a slower response. In addition, the delay of the first cAMP peak seen in Figure 6 compared with Figure 5 (1 h vs. 20 min) is likely the result of other molecules secreted by macrophages in the co-culture system, because macrophages can secrete a variety of growth factors that may antagonize the effect of PGE1 . Interestingly, the secondary phase of the intracellular cAMP response was blocked by cycloheximide treatment (Fig. 6). This indicates that the secondary phase of cAMP elevation in SMC requires the synthesis of new proteins.
ACKNOWLEDGMENTS
FOOTNOTES
-linolenic acid; EIA, enzyme immunoassay; FBS, fetal bovine serum; FP, fish oil and primrose oil mix at a ratio of 1:3 (wt/wt); GLA, -linolenic acid; GLA-mix, safflower oil ethyl esters and purified GLA free fatty acid mix at a ratio of 65:35 (wt/wt); GLA-TG, GLA-enriched triglyceride; PDGF, platelet-derived growth factor; PG, prostaglandin; PKA, protein kinase A; PO, primrose oil; SMC, smooth muscle cell.
Manuscript received 3 March 1997. Initial reviews completed 16 April 1997. Revision accepted 23 May 1997.
LITERATURE CITED
-linolenic acid modulates macrophage-vascular smooth muscle cell interactions: evidence for a macrophage-derived soluble factor that downregulates DNA synthesis in smooth muscle cells.
Arterioscler. Thromb. Vasc. Biol.
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15:1397-1403
1B -adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch.
In Vitro Cell Dev. Biol.
1995;
31:595-600-linolenic acid-enriched oils on human polymorphonuclear-neutrophil biosynthesis of leukotriene B4 .
Am. J. Clin. Nutr.
1992;
55:39-45
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