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Articles

Dietary -Linolenic Acid Suppresses Aortic Smooth Muscle Cell Proliferation and Modifies Atherosclerotic Lesions in Apolipoprotein E Knockout Mice¹

Yang-Yi Fan^*, Kenneth S. Ramos^,** and Robert S. Chapkin^*,2

^* Molecular and Cell Biology Section, Faculty of Nutrition, Center for Environmental and Rural Health and ^** Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77840

²To whom correspondence should be addressed at 442 Kleberg Biotechnology Center, 2471 TAMU, Texas A & M University, College Station, TX 77843-2471. E-mail: r-chapkin{at}tamu.edu

	ABSTRACT

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The present study was conducted to evaluate the antiatherogenic effects of dietary -linolenic acid (GLA) (primrose oil) in apolipoprotein E (apoE) genetic knockout mice. Five-wk-old male mice were fed cholesterol-free diets containing 10 g/100 g lipid as corn oil (CO) [control diet, 0 mol/100 mol GLA and (n-3) polyunsaturated fatty acids (PUFA)], primrose oil (PO, 10 mol/100 mol GLA), fish oil-CO mix [FC; 9:1 wt/wt, 0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish oil-PO mix [FP, 1:3 wt/wt, 8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA] for 15 wk. Subsequently, diets were supplemented with cholesterol (1.25 g/100 g) and sodium cholate (0.5 g/100 g) and fed for an additional 10 and 16 wk. Plasma cholesterol and triglyceride levels generally did not differ among groups at 20, 30 and 36 wk of age. Mice fed GLA-containing diets (PO and FP) had significantly (P < 0.05) higher liver phospholipid levels of dihomo--linolenic acid, the elongated product of GLA, relative to CO and FC groups. Consumption of GLA (PO and FP diets) significantly reduced (P < 0.05) aortic vessel wall medial layer thickness at 20 and 30 wk. A parallel GLA-dependent suppression in the number of proliferating (proliferating cell nuclear antigen positive) aortic smooth muscle cells was also observed. Diets containing either GLA or (n-3) PUFA reduced (P < 0.05) atherosclerotic lesion size in 30-wk-old mice. These results indicate that dietary GLA can suppress smooth muscle cell proliferation in vivo and retard the development of diet-induced atherosclerosis in apoE knockout mice.

KEY WORDS: • gamma-linolenic acid • fish oil • aortic smooth muscle cells • apoE knockout mouse • atherosclerosis.

	INTRODUCTION

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Atherosclerosis is responsible for 50% mortality rates in the United States, Japan and Europe (1) . Atherosclerotic lesions result from an excessive inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle layers of the arterial wall (1) . A large number of growth regulatory factors participate in the pathogenesis of this disorder, with diet recognized as one of the most prevalent risk factors for disease occurrence. Recently, it was demonstrated that dietary supplementation with (n-3) polyunsaturated fatty acids (PUFA)³ mitigates the course of the inflammatory-fibroproliferative response associated with atherosclerosis in humans (2 3 4) . The role of other dietary lipids and the possible beneficial effect of select novel dietary fatty acids have not been adequately studied. As the current diet-lipid-heart hypothesis may be overly simplistic (5) , research must be directed toward a better understanding of the association between dietary fat intake and coronary disease.

The major objective of our research has been to evaluate the antiatherogenic potential of dietary -linolenic acid [GLA, 18:3(n-6)]. Natural sources of GLA include plant seed oils of evening primrose oil, which contain 8–10 g GLA/100 g, blackcurrant (16–17 g/100 g), borage (24–25 g/100 g) and fungal oils, such as Mucor javanicus (16–19 g/100 g) (6) . Our previous studies demonstrated that dietary GLA is rapidly elongated in vitro and in vivo to dihomo--linolenic acid [DGLA, 20:3(n-6)] in murine macrophages (7 8 9) . The elongated product, DGLA, can enhance production of macrophage-derived prostaglandin E₁ (PGE₁) (9 10 11) . This is noteworthy because PGE₁ is a potent inhibitor of vascular smooth muscle cell (SMC) proliferation (12) . Using a mouse peritoneal macrophage-aortic SMC coculture model, we demonstrated that dietary GLA generates a macrophage phenotype that inhibits SMC proliferation (11 ,13) . Although GLA has no direct effect on vascular SMC proliferation, a macrophage-derived soluble factor, PGE₁, appears to mediate the growth inhibitory response (11 ,14) . Because alteration of SMC proliferation is a pivotal factor implicated in the pathogenesis of atherosclerotic vessel disease (1) , an evaluation of the effect of dietary GLA on lesion development and progression of atherosclerosis in vivo is warranted.

Environmental variation and genetic heterogeneity have hampered human studies to identify possible genetic determinants of dietary responsiveness. The recent advancement of mouse models offers the potential for in-depth investigation of complex gene/diet interactions in the presence of one or more risk factors for atherosclerosis. apoE knockout (KO) mice were created by homologous recombination in embryonic stem cells (15) . The progressive series of atherosclerotic lesions that develop in these mice are similar to those found in humans (16) . Therefore, apoE KO mice represent an important genetic model for studying the atherosclerotic process and the biology of vascular SMC, the major reactive cell type in atherosclerosis (1 ,16) .

We hypothesized that dietary GLA can favorably modulate the atherogenic process by down-modulation of aortic SMC proliferation in vivo. Therefore, we used the apoE KO mouse model to evaluate the effect of dietary GLA on aortic vessel wall medial layer thickness, the number of proliferating [proliferating cell nuclear antigen (PCNA) positive] aortic SMC in the vessel wall and aortic lesion progression. Because dietary supplementation with (n-3) PUFA has been implicated in the reduction of atherosclerosis in humans and mice (2 ,3 ,4 ,17) , we also examined the combined effects of GLA and (n-3) PUFA on these variables. Evidence is presented here that GLA down-modulates aortic SMC proliferation and retards atherosclerotic lesion progression in vivo.

	MATERIALS AND METHODS

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Animal and diets.

All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of Texas A & M University. Five-wk-old male apoE KO mice (C57BL/6J-apoE, 10th generation) were purchased from Jackson Laboratories (Bar Harbor, ME). The apoE (-/-) status of the mice was confirmed by Southern blot analysis of tail genomic DNA (18) . All mice expressed a 3.0-kb fragment of pJPB63 when digested with BglII and a 2.4-kb fragment of pJPB63 when digested with EcoRI, which is consistent with an apoE (-/-) genotype (15) .

Five-wk-old mice were fed cholesterol-free diets containing 10 g/100 g lipid that consisted of corn oil (CO) [control diet, 0 mol/100 mol GLA or (n-3) PUFA], primrose oil (PO) (10 mol/100 mol GLA), fish/corn oil mix (FC, 9:1, wt/wt) [0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish/primrose oil mix (FP, 1:3, wt/wt) [8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA] as previously described (11) for 15 wk. The fatty acid composition of the diets has been published (11) . Diets also contained 20.0 g casein, 0.3 g DL-methionine, 44.0 g sucrose, 14.98 g cornstarch, 6.0 g cellulose, 3.5 g AIN-76 mineral mix, 1.0 g AIN-76 vitamin mix, 0.2 g choline chloride and 0.02 g t-butylhydroquinone per 100 g as previously described (19) . The 15-wk regimen was followed by feeding the same four diets supplemented with cholesterol (1.25 g/100 g) and sodium cholate (0.5 g/100 g) for an additional 10 wk (30-wk-old mice, 10 mice/diet) or 16 wk (36-wk-old mice, 5 mice/diet) period. This dietary regimen induces an extraordinary lipemia resulting in atheroma formation (20 ,21) . Semipurified diets were adequate in all nutrients and varied only in the dietary oil composition (19) . Antioxidant (vitamin E) levels in the diets were 73.8, 72.9, 175.0 and 101.2 IU/kg for CO, PO, FC and FP, respectively. Peroxide levels in the dietary oils were <5 mEq/kg (11) . Powdered diets were mixed every month, distributed into plastic Zip-lock bags, flushed with nitrogen and stored at -20°C. As part of 2 separate feeding studies, fresh food was provided daily, and mice consumed it ad libitum. Body weights were recorded weekly.

Tissue fixation.

The apoE KO mice (20 wk old, fed cholesterol-free diets for 15 wk; 30 wk old, fed cholesterol-free diet for 15 wk followed by cholesterol-enriched diets for 10 wk; 36 wk old, fed cholesterol-free diets for 15 wk plus cholesterol-enriched diet for 16 wk) were deprived of food overnight before they were killed at the end of each feeding period (22) . Mice were sedated by intraperitoneal injection of pentobarbital (80 mg/kg body). The chest and abdomen were opened, blood was drawn from the left ventricle and the heart and aorta were perfused with 4% paraformaldehyde (22 ,23) . After perfusion, the aorta was separated from the heart at the proximal portion of the ascending aorta and cut transversely at the distal end of the thoracic aorta (22 ,24) . This region was chosen because the aortic arch is the initial site of lesion formation in this animal model (23) . The liver was also removed, snap-frozen in liquid nitrogen and stored at -80°C for subsequent biochemical analyses. The thoracic aorta was continuously fixed at 4% paraformaldehyde for an additional 4 hr at 4°C. This was followed by serial ethanol washing (25) . The thoracic aorta was evenly divided into three parts and processed for paraffin embedding. Serial tissue cross sections (5 µm thick) were then prepared from the proximal end of each of the three parts. For each animal, nine cross sections (three consecutive cross sections from each of three parts) were used for lesion size and vessel wall medial layer thickness measurement.

Plasma levels of total cholesterol and triglyceride were determined by an enzymatic calorimetric method using an Hitachi 911 autoanalyzer (Texas A&M Veterinary Diagnostic Lab, College Station, TX) (26) .

Fatty acid analysis.

Livers were thawed on ice, homogenized in 0.1 mol potassium chloride/L and extracted according to the method of Folch as previously described (27) . Total phospholipid was separated by thin-layer chromatography on silica gel 60 plates in the solvent system of chloroform/methanol/acetic acid/water (90:8:1:0.8, v/v). Fatty acids in the total phospholipid fraction were transmethylated, and the resultant fatty acid methyl esters were analyzed by gas chromatography (27) .

Morphological observations.

Paraformaldehyde-fixed, paraffin-embedded aorta cross sections were cut (5 µm thickness) and stained with hematoxylin and eosin for light microscopy. Images were captured digitally using a Sony 3CCD color video camera linked to a Reichert Microstar IV light microscope. Lesion size and vessel wall medial layer thickness were measured by automated pixel counting using a computer-aided NIH Image analysis system, version 1.61 (28 ,29) . The edge of each lesion was traced using an automated feature of the software to determine size for each aortic section (30) . For vessel wall medial layer thickness measurement, 40 random, noncontiguous microscopic fields (10 fields per quarter section) were examined from each aortic cross section. All measures were performed on each section (40 fields total) and averaged to obtain the average vessel wall medial layer thickness (31) .

Immunohistochemistry.

PCNA, a marker of cell proliferation, was detected using antibody PC-10 (Dako) (80 mg/L). Anti–smooth muscle -actin antibody (Sigma Chemical Co., St. Louis, MO) (20 mg/L) and anti-mouse macrophage antibody (Accurate) (50 mg/L) were used to identify SMC and macrophages, respectively. For immunostaining, endogenous peroxidase activity was blocked by incubating sections with 3% H₂O₂ in methanol. Anti–PC-10 was labeled with biotin and applied to the sections, followed by avidin-biotin amplification. Antibody binding was visualized with diaminobenzidine (32 ,33) . For cell-type identification, biotinylated anti–smooth muscle -actin antibody or anti-mouse macrophage antibody was applied to each section, followed by streptavidin-alkaline phosphatase treatment and development with alkaline phosphatase substrate (34 ,35) . The percentage of proliferating SMC was calculated by dividing the number of PCNA-positive SMC by the total number of SMC in the entire aortic medial layer vessel wall.

Statistical analysis.

Differences among dietary groups were analyzed by one-way ANOVA. Post hoc analyses of significance were made by Scheffé’s test. Differences with P < 0.05 were accepted as significant.

	RESULTS

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Growth and serum lipids.

No significant differences (P > 0.05) in body weight among the diet groups were observed at any time point. The final body weights for 36-wk-old mice were 26.8 ± 0.9 g for CO, 27.1 ± 0.1 for PO, 27.1 ± 0.5 for FC, and 29.4 ± 0.4 for FP (n = 4), indicating that test dietary lipids resulted in comparable growth rates. Neither dietary GLA nor (n-3) PUFA significantly affected (P > 0.05) plasma lipid concentrations (Table 1 ) with one exception; at 36 wk, there was a significant elevation in serum triglyceride concentrations in mice fed FC relative to controls fed the CO diet. In all dietary groups, mice fed cholesterol-enriched diets (30- and 36-wk-old mice) had higher serum cholesterol levels than those fed cholesterol-free diets.

At 30 wk, relative to the control CO group, PO- and FP-fed mice had significantly (P < 0.05) elevated levels of DGLA (Table 2 ), indicating that on incorporation of dietary lipid in vivo, GLA was metabolically elongated to DGLA. In addition, (n-3) PUFA-enriched FC and FP groups had higher (P < 0.05) levels of eicosapentaenoic acid (EPA) [EPA, c20:5(n-3)] and docosahexaenoic acid (DHA) [DHA, c22:6(n-3)] and lower arachidonic acid [c20:4(n-6)] compared with CO and PO mice.

Mice fed diets containing GLA (PO and FP) had significantly (P < 0.05) lower vessel wall thickness compared with CO- and FC-fed mice at 20 and 30 wk of age (Fig. 1 ). Because proliferation of SMC constitutes a key event in medial layer thickening and progression of atherosclerosis (1) , we next examined the percentage of proliferating SMC in the thoracic aorta vessel wall medial layer. In general, total SMC number in the aortic medial layer vessel wall (cross section) ranged from 350 to 550 cells (460 ± 25, means ± SE, n = 20) in areas where there was minor or no lesion development. However, SMC number increased to >1377 ± 276 (n = 20) at the site of lesion formation. Mice fed GLA (PO and FP groups) had significantly fewer (P < 0.05) proliferating (PCNA-positive) SMC than rats fed CO and FC [percent proliferating SMC (n = 5) in PO, FP, CO and FC were 4.34 ± 0.55, 3.55 ± 1.07, 10.53 ± 2.07 and 7.50 ± 0.79, respectively]. These effects were most pronounced at 30 wk of age, with similar but less pronounced effects at 36 wk of age (Fig. 2 ). FC treatment had no effect (P > 0.05) on vessel wall medial thickness or PCNA-positive SMC at 30 wk compared with CO-fed mice, indicating that dietary vitamin E levels were not related to alterations in SMC proliferative status.

View larger version (21K): [in this window] [in a new window]   Figure 1. Quantitative analysis of thoracic wall medial layer thickness in apolipoprotein E (apoE) knockout (KO) mice fed different dietary lipids. Five-wk-old male apoE KO mice were initially fed cholesterol-free diets (10 g/100 g) containing either corn oil (CO) [control diet, 0 mol/100 mol -linolenic acid (GLA) or (n-3) polyunsaturated fatty acids (PUFA)], primrose oil (PO) [10 mol/100 mol GLA], fish/corn oil mix (FC, 9:1, wt/wt) [0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish/primrose oil mix (FP, 1:3, wt/wt) [8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA] for 15 wk, followed by feeding the same diets supplemented with cholesterol (1.25 g/100 g) and sodium cholate (0.5 g/100 g) for an additional 10- or 16-wk period. Thoracic aortas were isolated from apoE KO mice fed experimental diets as described in the text. Microscopic images of hematoxylin and eosin–stained aorta cross sections were captured digitally and quantified as described in the text. Average vessel wall medial layer thickness is the average of 40 random, noncontiguous microscopic fields (10 fields per quarter section) measured from each aortic cross section. Data represent means ± SE, n = 4–9 mice per time point from two separate feeding experiments. *Significantly different (P < 0.05) from control (CO) mice.

View larger version (17K): [in this window] [in a new window]   Figure 2. Percentage of proliferating cell nuclear antigen (PCNA)-positive smooth muscle cells (SMC) in thoracic aorta vessel wall medial layer of 30- and 36-wk-old apolipoprotein E (apoE) knockout (KO) mice fed corn oil (CO) [control diet, 0 mol/100 mol -linolenic acid (GLA) or (n-3) polyunsaturated fatty acids (PUFA)], primrose oil (PO) [10 mol/100 mol GLA], fish/corn oil mix (FC, 9:1, wt/wt) [0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish/primrose oil mix (FP, 1:3, wt/wt) [8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA]. Paraformaldehyde-fixed, paraffin-embedded aorta cross sections were immunostained with PCNA antibody PC-10 (Dako) as described in the text. The percentage of proliferating SMC was calculated by dividing the total number of PCNA-positive SMC by the total number of SMC in the entire aortic medial layer vessel wall. Data represent means ± SE, n = 5 mice per treatment from two separate feeding experiments. Values sharing a superscript are not significantly different (P > 0.05).

No major lesions were found in the thoracic aortas in 20-wk-old mice for any of the cholesterol-free diets tested (Fig. 3A ), indicating that disease progression is dependent on exposure to dietary cholesterol. After the addition of cholesterol (1.25 g/100 g) and sodium cholate (0.5 g/100 g) for an additional 10- or 16-wk feeding period, mice from all dietary groups developed atherosclerotic lesions (Fig. 3A) . Representative cross sections are shown in Figs. 3B and 3C . The lesions ranged from multilayered foam cell deposits to fibrous and advanced plaques. This is consistent with previous reports indicating that apoE KO mice develop the entire spectrum of lesion types, similar to humans (16) . Although we did not evaluate the effect of dietary lipid on lesion type, immunohistochemical staining indicated that PCNA-positive macrophages were primarily localized to the intimal lesion area. In agreement with previous reports (35 36 37) , SMC were predominantly located in the medial layer and in the periphery of atherosclerotic plaques (data not shown). GLA and (n-3) PUFA reduced aortic lesion size at 30 wk of age, 10 wk after the initiation of cholesterol feeding (Fig. 3) . Specifically, mice fed CO developed larger atherosclerotic lesions than the PO, FC and FP groups. By 36 wk of age, all mice developed large lesions, and no diet-related effects could be discerned.

View larger version (85K): [in this window] [in a new window]   Figure 3. Lesion progression in apolipoprotein E (apoE) knockout (KO) mice fed corn oil (CO) [control diet, 0 mol/100 mol -linolenic acid (GLA) or (n-3) polyunsaturated fatty acids (PUFA)], primrose oil (PO) [10 mol/100 mol GLA], fish/corn oil mix (FC, 9:1, wt/wt) [0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish/primrose oil mix (FP, 1:3, wt/wt) [8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA]. Paraffin-embedded thoracic aorta sections from 20-, 30- and 36-wk-old mice were stained with hematoxylin and eosin, and images of aorta cross sections were captured as described in the text. A, Lesion size was measured by automated pixel counting using a computer-aided NIH Image analysis system (version 1.61). Data represent means ± SE, n = 4–9 mice per time point from two separate feeding experiments. *Significantly different (P < 0.05) from CO. Representative cross sections of the thoracic aorta from 30- (B) and 36- (C)-wk-old mice fed different dietary lipids (original magnification 100x). Arrow indicates lesion.

	DISCUSSION

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apo E KO mice develop lesions similar to humans as a function of dietary fat content and therefore are recognized as an important animal model for studying the effect of dietary lipids on atherosclerosis (16) . In support of our hypothesis, mice fed diets containing GLA had reduced medial layer thickness of the aortic wall. This response was in part due to reduced mitotic activity of SMC within the aortic wall. PGE₁ is a possible candidate factor responsible for the down-regulation of SMC proliferation by GLA (12 ,37) . Although we did not measure the prostaglandin profile of the aorta, tissue levels of DGLA, a precursor to PGE₁, were significantly (P < 0.05) enhanced after GLA feeding (Table 2) .

The progressive series of atherosclerotic lesions that develop in the apoE KO mouse were lower in PO, FC and FP groups compared with the control (CO) diet–fed mice during the intermediate stages of disease progression (30 wk of age). The lack of an effect of either GLA or (n-3) PUFA at 36 wk of age likely reflects the overwhelming hyperlipidemia observed in the apoE KO mouse, making it difficult to detect effects of dietary lipids at these late time points (15 ,41) . However, because only a single dose of GLA was used in these studies, it is possible that the selected level was above or possibly below the beneficial pharmacology for a long-term study. Interestingly, no major lesions were found in the thoracic aortas at 20 wk of age, before the introduction of dietary cholesterol. Although en face techniques may be more sensitive for the purpose of quantifying small intimal lesions, this assay precludes the analysis of relevant atherosclerotic biomarkers in the same tissue. Therefore, to perform additional immunohistochemical analyses (e.g., PCNA and cell-type localization), we measured lesion size on aorta cross sections as previously described (42) . The aortic arch was selected because previous studies of apoE KO mice have demonstrated that the proximal aorta is especially prone to atherosclerosis (43 44 45) .

The lack of major lesion development in the absence of cholesterol indicates that dietary cholesterol is the primary factor driving atherosclerotic lesion development in apoE KO mice. This finding has often been overlooked by researchers studying the atherogenic response of apo E KO mice fed commercially available nonpurified diet, containing 0.03 g cholesterol/100 g. The dependence of aortic lesion development on dietary cholesterol is consistent with previous studies, indicating its requirement for driving lesion development (46 ,47) . In addition, although cholesterol + sodium cholate supplementation increased plasma cholesterol levels, dietary lipid sources did not further influence plasma total cholesterol or triglyceride levels, indicating that dietary GLA and (n-3) PUFA can retard lesion formation independent of plasma lipid levels.

Although lipid oxidation plays an important role in atherogenesis, the inhibitory effect of antioxidants on atherogenesis in apoE KO mice is controversial (48 49 50 51) . Because no correlation between dietary vitamin E levels and alterations in SMC proliferation was observed, it is unlikely that dietary GLA influenced SMC susceptibility to oxidative injury (50) . Although the FC diet contained the highest level of vitamin E among the four dietary groups, it had no effect on vascular medial layer thickness or the percentage of PCNA positive cells compared with the control diet (CO), suggesting that dietary vitamin E was not a contributing factor to the retardation of aortic SMC proliferation and lesion development observed in our study. In addition, because the linoleic acid [18:2(n-6)] contents of the CO and PO diets were similar (11) , the protective effects of PO cannot be attributed to general effects of (n-6) PUFA (21) .

Despite the fact that dietary (n-3) PUFA can retard atherosclerotic lesion development in apoE KO mice, the protective effect was not due to the inhibition of SMC proliferation, as shown in Figs. 1 and 2 . This observation is consistent with a previous in vitro macrophage-SMC coculture study (11) where (n-3) PUFA-enriched macrophages did not significantly inhibit SMC proliferation in the absence of zymosan stimulation. Because (n-3) PUFA (EPA and DHA) and (n-6) PUFA compete for access to receptors and certain enzymes (e.g., cyclooxygenases, lipoxygenases), the enrichment of (n-3) PUFA in vivo (Table 2) may inhibit the production of arachidonic acid–derived proinflammatory metabolites, resulting in retardation of lesion development. Further studies are required to elucidate the distinct mechanisms by which GLA and (n-3) PUFA ameliorate the atherogenic process.

In conclusion, this study demonstrates that GLA-containing PO reduces SMC proliferation within the aortic wall and modifies atherosclerotic lesion progression. The antiatherogenic effect of GLA is not accounted for by its effect on serum lipids or the levels of vitamin E in the diet. Further experiments are required to elucidate the mechanism(s) by which dietary GLA down-regulates vascular SMC growth in vivo. It is possible that the GLA-derived cyclooxygenase product PGE₁ is responsible for the antiproliferative dietary effects. This particular research focus may lead to a better understanding of the precise role of specific (n-6) and (n-3) PUFA in the pathogenesis of atherosclerosis.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the National Institutes of Health Fish Oil Test Material Program for providing the vacuum deodorized fish oil. The assistance of Jorge Piedrahita (Texas A & M University) with apoE (-/-) genotype identification is also greatly appreciated.

	FOOTNOTES

 
¹ Supported in part by a grant from the Texas A & M Interdisciplinary Research Enhancement Program, Traco Labs and the National Institute of Environmental Health Sciences (Grant P30-ES-09106).

³ Abbreviations used: apoE, apolipoprotein E; CO, corn oil; DGLA, dihomo--linolenic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FC, fish oil-CO mix; FP, fish oil-PO mix; GLA, -linolenic acid; KO, knockout; PCNA, proliferating cell nuclear antigen; PGE₁, prostaglandin E₁; PO, primrose oil; PUFA, polyunsaturated fatty acids; SMC, smooth muscle cell.

Manuscript received December 6, 2000. Initial review completed February 7, 2001. Revision accepted March 12, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Ross R. Cell biology of atherosclerosis. Annu. Rev. Physiol. 1995;57:791-804[Medline]

2. Daviglus M. L., Stamler J., Orencia A., Dyer A. R., Liu K. L., Greenland P., Walsh M. K., Morris D., Shekelle R. B. Fish consumption and the 30-year risk of fatal myocardial infarction. N. Engl. J. Med. 1997;336:1046-1053[Abstract/Free Full Text]

3. Marchioli R. Dietary supplementation with (n-3) polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Lancet 1999;354:447-455[Medline]

4. Von Schacky C., Angerer P., Kothny W., Theisen K., Mudra H. The effect of dietary -3 fatty acids on coronary atherosclerosis. a randomized, double blind, placebo-controlled trial. Ann. Intern. Med. 1999;130:554-562[Abstract/Free Full Text]

5. Esrey K. L., Joseph L., Grover S. A., Jr Relationship between dietary intake and coronary heart disease mortality: Lipid Research Clinics prevalence follow-up study. Clin. Epidemol. 1996;49:211-216

6. Lawson L. D., Hughes B. G. Triacylglycerol structure of plant and fungal oils containing -linolenic acid. Lipids 1988;23:313-317

7. Chapkin R. S., Somer S. D., Erickson K. L. Dietary manipulation of macrophage phospholipid classes: selective increase of dihomogammalinolenic acid. Lipids 1988;23:766-770[Medline]

8. Chapkin R. S., Miller C. C., Somers S. D., Erickson K. L. Utilization of dihomogammalinolenic acid (8,11,14-eicosatrienoic acid) by murine peritoneal macrophages. Biochim. Biophys. Acta 1988;959:322-331[Medline]

9. Fan Y-, Y. & Chapkin R. S. Enhancement of mouse peritoneal macrophage prostaglandin E₁ by dietary gamma-linolenic acid. J. Nutr. 1992;122:1600-1606

10. Fan Y-, Y. & Chapkin R. S. Phospholipid sources of metabolically elongated gammalinolenic acid: conversion to prostaglandin E₁ in stimulated mouse macrophages. J. Nutr. Biochem. 1993;4:602-607

11. Fan Y-Y., Ramos K. S., Chapkin R. S. Dietary -linolenic acid modulates macrophage-vascular smooth muscle cell interactions. Arterioscler. Thromb. Vasc. Biol. 1995;15:1397-1403[Abstract/Free Full Text]

12. Fan Y-Y., Ramos K. S., Chapkin R. S. Cell cycle-dependent inhibition of DNA synthesis in vascular smooth muscle cells by prostaglandin E₁: relationship to intracellular cAMP levels. Prost. Leuk. Essential Fatty Acids 1996;54:101-107

13. Fan Y-Y., Chapkin R. S., Ramos K. S. A macrophage-smooth muscle cell co-culture model: applications in the study of atherogenesis. In Vitro Cell. Dev. Biol. 1995;31:492-493

14. Fan Y-Y., Chapkin R. S., Ramos K. S. Dietary gamma-linolenic acid enhances release of macrophage-derived prostaglandin E₁ leading to inhibition of mouse vascular smooth muscle cell proliferation. J. Nutr. 1997;127:1765-1771[Abstract/Free Full Text]

15. Piedrahita J. A., Zhang S. H., Hagaman J. R., Oliver P. M., Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Sci. U S A. 1992;89:4471-4475[Abstract/Free Full Text]

16. Breslow J. L. Mouse models of atherosclerosis. Science 1996;272:685-688[Abstract]

17. Renier G., Skamene E., DeSanctis J., Radzioch D. Dietary (n-3) polyunsaturated fatty acids prevent the development of atherosclerotic lesions in mice: modulation of macrophage secretory activities. Atheroscler. Thromb. 1993;13:1515-1524[Abstract/Free Full Text]

18. Murray N. R., Davidson L. A., Chapkin R. S., Gustafson W. C., Schattenberg D. G., Fields A. P. Overexpression of protein kinase C beta II in the colonic epithelium causes hyperproliferation and increased sensitivity to colon carcinogenesis. J. Cell Biol. 1999;145:699-711[Abstract/Free Full Text]

19. Fan Y-Y., Chapkin R. S., Ramos K. S. Dietary lipid source alters murine macrophage/vascular smooth muscle cell interactions in vitro. J. Nutr. 1996;126:2083-2088

20. Black T. M., Wang P., Maeda N., Coleman R. A. Palm tocotrienols protect apoE +/- mice from diet-induced atheroma formation. J. Nutr. 2000;130:2420-2426[Abstract/Free Full Text]

21. George J., Mulkins M., Casey S., Schatzman R., Sigal E., Harats D. The effects on (n-6) polyunsaturated fatty acid supplementation on the lipid composition and atherosclerosis in mouse models of atherosclerosis. Atherosclerosis 2000;150:285-293[Medline]

22. Reddick R. L., Zhang S. H., Maeda N. Atherosclerosis in mice lacking apo E: evaluation of lesional development and progression. Arterioscler. Thromb. 1994;14:141-147[Abstract/Free Full Text]

23. Ni W., Tsuda Y., Sakono M., Imaizumi K. Dietary soy protein isolate, compared with casein, reduces atherosclerotic lesion area in apolipoprotein E-deficient mice. J. Nutr. 1998;128:1884-1889[Abstract/Free Full Text]

24. Nakashima Y., Plump A. S., Raines E. W., Brestow J. L., Ross R. apoE-deficient mice develop lesions during phases of atherosclerosis throughout the arterial tree. Arterioscler. Thromb. 1994;14:133-139[Abstract/Free Full Text]

25. Jiang Y. H., Aukema H. M., Lupton J. R., Chapkin R. S. Localization of protein kinase C isozymes in rat colon. Cell Growth Differ 1995;6:1381-1386[Abstract]

26. Crawford R. S., Kirk E. A., Rosenfeld M. E., LeBoeuf R. C., Chait A. Dietary antioxidants inhibit development of fatty streak lesions in the LDL receptor-deficient mouse. Arterioscler. Thromb. Vasc. Biol. 1998;18:1506-1513[Abstract/Free Full Text]

27. Chapkin R. S., Coble K. J. Remodeling of mouse kidney phospholipid classes and subclasses by diet. J. Nutr. Biochem. 1991;2:158-164

28. Fazio S., Sanan D. A., Lee Y. L., Ji Z. S., Mahley R. W., Rall S. C., Jr Susceptibility to diet-induced atherosclerosis in transgenic mice expressing a dysfunctional human apolipoprotein E (Arg 112, Cys 142). Arterioscler. Thromb. 1994;14:1873-1879[Abstract/Free Full Text]

29. Hong M. Y., Chapkin R. S., Morris J. S., Wang N., Carroll R. J., Turner N. D., Lupton J. R. Relationship between DNA adduct levels, repair enzyme and apoptosis as a function of DNA alkylation by azoxymethane. Cell Growth Differ 1999;10:749-758[Abstract/Free Full Text]

30. Tangirala R. K., Rubin E. M., Palinski W. Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J. Lipid Res. 1995;36:2320-2328[Abstract]

31. Capron L., Jarnet L., Heudea D., Joseph-Monrose D., Bruneval P. Repeated balloon injury of rat aorta: a model of neointima with attenuated inhibition by heparin. Arterioscler Thromb. Vasc. Biol. 1997;17:1649-1656[Abstract/Free Full Text]

32. Zeymer U., Fishbein M. C., Forrester J. S., Cercek B. Proliferating cell nuclear antigen immunohistochemistry in rat aorta after balloon denudation. Am. J. Pathol. 1992;141:685-690[Abstract]

33. Hierck B. P., Iperen L. V., Gittenberger-de Groot A. C., Poelmann R. E. Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies. J. Histochem. Cytochem. 1994;42:1499-1502[Abstract]

34. Palinski W., Ord V. A., Plump A. S., Breslow J. L., Steinberg D., Witztum J. L. Apo E-deficient mice are a model of lipoprotein oxidation in atherogenesis: demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler. Thromb. Vasc. Biol. 1994;14:605-616[Abstract/Free Full Text]

35. Rekhter M. D., Gordon D. Active proliferation of different cell types, including lymphocytes, in human atherosclerotic plaques. Am. J. Pathol. 1995;147:668-677[Abstract]

36. Zhou X., Stemme S., Hansson G. K. Evidence for a local immune response in atherosclerosis: CD4⁺ T cells infiltrate lesions of apolipoprotein-E deficient mice. Am. J. Pathol. 1996;149:359-366[Abstract]

37. Fan Y-Y., Chapkin R. S. Importance of dietary -linolenic acid in human health and nutrition. J. Nutr. 1998;128:1411-1414[Abstract/Free Full Text]

38. Sassen L., Lamers J., Sluiter W., Hartog J., Dekkers D., Hogendoorn A., Verdouw P. Development of regression of atherosclerosis in pigs: effects of (n-3) fatty acids, their incorporation into plasma and aortic plaque lipids, and granulocyte function. Arterioscler. Thromb. 1993;13:651-660[Abstract/Free Full Text]

39. Stone N. J. Fish consumption, fish oil, lipids, and coronary heart disease. Am. J. Clin. Nutr. 1997;65:1083-1086[Free Full Text]

40. Billman G., Jing X., Leag A. Prevention of sudden cardiac death by dietary pure -3 polyunsaturated fatty acids in dogs. Circulation 1999;99:2452-2457[Abstract/Free Full Text]

41. Plump S., Smith J. D., Hayek T., Aalto-Setala K., Walsh A., Verstuyft J. G., Rubin E. M., Breslow J. L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 1992;71:343-353[Medline]

42. Moghadasian M. H., McManus B. M., Pritchard P. H., Frohlich J J. "Tail oil"-derived phytosterols reduce atherosclerosis in apoE deficient mice. Arterioscler. Thromb. Vasc. Biol. 1997;17:119-126[Abstract/Free Full Text]

43. Caliguiuri G., Nicoletti A., Zhou X., Tornberg I., Hansson G. K. Effects of sex and age on atherosclerosis and autoimmunity in apoE-deficient mice. Atherosclerosis 1999;145:301-308[Medline]

44. Aviram M, Dornfeld L., Rosenblat M., Volkova N., Kaplan M., Coleman R., Hayek T., Presser D., Fuhrman B. Pomegranate juice consumption reduces oxidative stress atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am. J. Clin. Nutr. 2000;71:1062-1076[Abstract/Free Full Text]

45. Grimsditch D. C., Penfold S., Latcham J., Vidgeon-Hart M., Groot P.H.E., Benson G. M. C3H apoE (-/-) mice have less atherosclerosis than C57BL apoE (-/-) mice despite having a more atherogenic serum lipid profile. Atherosclerosis 2000;151:389-397[Medline]

46. Calleja L., Paris M., Paul A., Vilella E., Joven J., Jimenez A., Beltran G., Uceda M., Maeda N., Osada J. Low-cholesterol and high-fat diets reduce atherosclerotic lesion development in apoE-knockout mice. Arterioscler. Thromb. Vasc. Biol. 1999;19:2368-2375[Abstract/Free Full Text]

47. Staprans I., Pan X., Rapp J., Grunfeld C., Feingold K. Oxidized cholesterol in the diet accelerates the development of atherosclerosis in LDL receptor- and apolipoprotein E–deficient mice. Arterioscler. Thromb. Vasc. Biol. 2000;20:708-714[Abstract/Free Full Text]

48. Zhang S. H., Reddick R. L., Avdievich E., Surles L. K., Jones R. G., Reynolds J. B., Quarfordt S. H., Maeda N. Paradoxical enhancement of atherosclerosis by probucol treatment in apolipoprotein E-deficient mice. J. Clin. Invest. 1997;99:2858-2866[Medline]

49. Pratico D., Tangirala R. K., Rader D. J., Rokach J., FitzGerald G. A. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in apo E-deficient mice. Nat. Med. 1998;4:1189-1192[Medline]

50. Shaish A., George J., Gilburd B., Keren P., Levkovitz H., Harats D. Dietary ß-carotene and -tocopherol combination does not inhibit atherogenesis in an apoE-deficient mouse model. Arterioscler Thromb Vasc Biol 1999;19:1470-1475[Abstract/Free Full Text]

51. Osada J., Joven J., Maeda N. The value of apolipoprotein E knockout mice for studying the effects of dietary fat and cholesterol on atherogenesis. Curr. Opin. Lipidol. 2000;11:25-29[Medline]

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