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Am J Physiol Heart Circ Physiol 291: H2987-H2996, 2006. First published July 14, 2006; doi:10.1152/ajpheart.01179.2005
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Effects of dietary flaxseed on vascular contractile function and atherosclerosis during prolonged hypercholesterolemia in rabbits

C. M. C. Dupasquier,1,2,3 A.-M. Weber,1,2,3 B. P. Ander,1,2,3 P. P. Rampersad,2,3 S. Steigerwald,1 J. T. Wigle,2,6 R. W. Mitchell,7 E. A. Kroeger,3 J. S. C. Gilchrist,1,2,3,4 M. M. Moghadasian,1,5 A. Lukas,2,3 and G. N. Pierce1,2,3

1Canadian Centre for Agri-Food Research in Health and Medicine, 2Institute for Cardiovascular Sciences, St. Boniface Hospital Research Centre, 3Departments of Physiology, 4Oral Biology, 5Human Nutritional Sciences, and 6Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada; and 7Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois

Submitted 8 November 2005 ; accepted in final form 4 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dietary flaxseed has significant anti-atherogenic effects. However, the limits of this action and its effects on vascular contractile function are not known. We evaluated the effects of flaxseed supplementation on atherosclerosis and vascular function under prolonged hypercholesterolemic conditions in New Zealand White rabbits assigned to one of four groups for 6, 8, or 16 wk of feeding: regular diet (RG), 10% flaxseed-supplemented diet (FX), 0.5% cholesterol-supplemented diet (CH), and 0.5% cholesterol- and 10% flaxseed-supplemented diet (CF). Cholesterol feeding resulted in elevated plasma cholesterol levels and the development of atherosclerosis. The CF group had significantly less atherosclerotic lesions in the aorta and carotid arteries after 6 and 8 wk than the CH animals. However, the anti-atherogenic effect of flaxseed supplementation was completely attenuated by 16 wk. Maximal tension induced in aortic rings either by KCl or norepinephrine was not impaired by dietary cholesterol until 16 wk. This functional impairment was not prevented by including flaxseed in the high-cholesterol diet. Aortic rings from the cholesterol-fed rabbits exhibited an impaired relaxation response to acetylcholine at all time points examined. Including flaxseed in the high-cholesterol diet completely normalized the relaxation response at 6 and 8 wk and partially restored it at 16 wk. No significant changes in the relaxation response induced by sodium nitroprusside were observed in any of the groups. In summary, dietary flaxseed is a valuable strategy to limit cholesterol-induced atherogenesis as well as abnormalities in endothelial-dependent vasorelaxation. However, these beneficial effects were attenuated during prolonged hypercholesterolemic conditions.

linseed; acetylcholine; nutrition; polyunsaturated fatty acids; vascular relaxation

ATHEROSCLEROSIS IS THE leading cause of cardiovascular morbidity and mortality in North America (77). Atherosclerosis induces two significant pathological processes: an ischemic event due to blood flow obstruction and vascular contractile dysfunction. It is well known that atherosclerosis is associated with elevated circulating cholesterol levels. Elevated plasma cholesterol concentrations induced by cholesterol feeding result in the development of atherosclerosis and an impairment in endothelium-dependent vasodilation in rabbits (9, 26, 29, 30, 36). The development of interventions to inhibit cholesterol-induced atherosclerosis and the associated vascular dysfunction have received much attention because of this strong association. For example, there is an increasing interest in nutritional interventions that may prevent the development of atherosclerosis and protect against the vascular function abnormalities induced by cholesterol consumption. Flaxseed is one such novel dietary intervention. Flaxseed is a good source of soluble and insoluble dietary fiber and is the richest plant source of {alpha}-linolenic acid [ALA; C18:3 n-3, omega-3 (n-3) fatty acid] as well as the lignan secoisolariciresinol diglucoside (SDG) (39, 53, 68). Whole ground flaxseed or the derivatized components of flaxseed have exhibited cardioprotective and anti-atherogenic properties both clinically (7, 8, 12, 32, 42, 47) and in several animal models (41, 52, 54, 56, 59, 62, 64, 71, 76). However, these results were observed using rather short periods of cholesterol feeding. The effects of a dietary intervention with flaxseed during prolonged periods of cholesterol supplementation are uncertain. More importantly, it is not known whether dietary flaxseed can prevent the negative effects on vascular function that are induced by cholesterol.

The objective of the present study was to determine the effects of dietary supplementation with flaxseed on vascular function and atherosclerotic lesion development during prolonged hypercholesterolemic conditions. We selected a rabbit model to test four different dietary conditions: a regular control diet, a flaxseed-supplemented diet, a diet containing elevated cholesterol levels, and a diet containing both cholesterol and flaxseed. We hypothesized that dietary flaxseed would limit atherosclerotic development and demonstrate a protective effect against cholesterol-induced vascular contractile abnormalities.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and dietary interventions. Ninety-six male albino New Zealand White (NZW) rabbits (Southern Rose Rabbitry Farm, St. Claude, Canada), weighing 2.5–3 kg on arrival, were individually housed in metal cages in a room with controlled temperature, humidity, and a 12-h light cycle. Experiments were reviewed and approved by the University of Manitoba Protocol Management Review Committee, in accordance with Canadian Council on Animal Care guidelines (48). Animals were randomly assigned to four groups of eight animals per feeding duration based on dietary treatment. Animals were fed for 6, 8, or 16 wk. The four diets included a control diet (RG) of regular rabbit chow (CO-OP Complete Rabbit Ration, Federal Cooperatives, Saskatoon, Canada), a 10% ground flaxseed-supplemented chow (FX), a 0.5% cholesterol-supplemented chow (CH), and a diet supplemented with 0.5% cholesterol and 10% ground flaxseed (CF). The Promega flaxseed, provided from Polar Foods in Fisher Branch, Canada, contained 71% ALA. All diets were dry mixed and repelleted to incorporate the added components. Rabbits were fed 125 g of the appropriate dietary treatment per day. Water was given ad libitum.

Blood sampling and analysis. Blood samples were taken at baseline (0 wk) and after 6, 8, or 16 wk of dietary intervention from the marginal ear vein before daily feeding. Plasma cholesterol and triglyceride levels were measured enzymatically using commercial kits (Thermo Electron). Total fatty acids were extracted from the plasma samples and derivatized as previously described (4, 38). Fatty acids were esterified into their corresponding methyl esters, using an acetylchloride-methanol-benzene solution. Subsequent analysis by gas chromatography (GC) with flame ionization detection (FID) yielded the amounts of fatty acid methyl esters (FAMEs) quantitatively. The fatty acid content of the samples was identified by comparison with authentic standards (NuChek Prep, Elysian, MN).

Preparation of tissues. After 6, 8, or 16 wk of dietary treatment, rabbits were anesthetized with isofluorane (5%, in oxygen, 2 l/min) and heparinized. The aorta and carotid arteries were excised and immediately placed in cold Krebs-Henseleit 1.9 mM calcium solution (115 mM NaCl, 25 mM NaHCO3, 1.38 mM KH2PO4, 2.5 mM KCl, 2.46 mM MgSO4, 1.9 mM CaCl2, 11.2 mM dextrose, pH 7.4). The aorta was carefully dissected from the distal end of the aortic arch to the base of the diaphragm. The aorta and carotids were cleaned of adventitial tissue and prepared for vascular function testing, GC, sectioning, or en face analysis.

Assessment of atherosclerotic lesion formation. Atherosclerotic lesions along the distal aorta and carotid artery were evaluated en face and by cross-sectional analysis. For en face analysis, the aorta and carotid arteries were cleaned of peripheral tissue, opened longitudinally, and digitally photographed, and the luminal images were analyzed using Silicon Graphics Imaging software. The lesion area was calculated as a percentage of the total luminal area covered by atherosclerotic lesions.

Aortic tissue, fixed in 4% buffered paraformaldehyde and rinsed with 30% sucrose solution buffered in 1x PBS, was embedded in tissue-freezing medium (optimal cutting temperature compound), frozen at –20°C, cut into 10-µm-thick sections using a cryostat, and thaw mounted onto positive glass slides. Sections were stained with Oil Red O and counterstained with hematoxylin as previously described (44).

Following en face atherosclerosis analysis, the aortas were stored at –80°C, thawed, and homogenized in preparation for chloroform-methanol lipid extraction as previously described in detail (4, 21). The extracted lipids were quantified as described above.

Experimental protocol for assessing vascular response. Aortic tissue, dissected into 3-mm-width rings from the distal end of the aortic arch, was fastened in an organ bath with surgical wire, perfused with the Krebs-Henseleit solution, aerated with 95% O2 and 5% CO2, and equilibrated at 37°C and pH 7.4. Vascular function was measured with a force transducer as mechanograms of tension [tension (g)/tissue wet wt (g)]. The aortic rings were brought to a basal tension of 5.5–6.5 g of tension and then contracted three times with 47 mM KCl, with washout periods using Krebs solution between each contraction. Tissues were allowed to return to baseline tension during washouts. A dose-response curve to norepinephrine (NE) was constructed with concentrations of 10–9 to 10–4 M. After the final dose of NE, the tissues were washed out with 37°C Krebs-Henseleit solution and allowed to return to baseline tension. To test the ability of the tissue to relax after precontraction with NE, a second dose of 10–6 M NE was administered to the bath, and the tissues were allowed to reach a steady state of contraction. Acetylcholine (ACh) was then administered without washout at concentrations of 10–8 to 10–5 M to develop a relaxation response curve to ACh. After washout and a third dose of 10–6 M NE, sodium nitroprusside (SNP) was administered in selected experiments at concentrations of 10–8 to 10–5 M to generate a relaxation response curve.

Statistical analyses. Data are expressed as means ± SE. Statistical comparisons were made using one-way ANOVA, followed by Fishers least significant difference test for multiple parametric comparisons. Differences between means were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Diet composition and animal weights. Animals in all four treatment groups consumed 125 g of chow daily. Animal body weights did not differ significantly among the four groups before feeding (0 wk) or at the end of the feeding trials (6, 8, or 16 wk) (data not shown), which suggests that the energy content of the experimental diets did not differ significantly. Data measuring the nutritional composition of the experimental diets support this contention and are reported elsewhere (4). The addition of 10% flaxseed notably elevated the total fat and ALA content of the FX and CF diets. The ratio of n-6 to n-3 polyunsaturated fatty acids (PUFA) was ninefold less in the flaxseed-supplemented diets compared with the RG and CH diets (4).

Effects on lipid concentrations. Initial plasma cholesterol and triglyceride levels were not significantly different among the four groups (Fig. 1). After 6, 8, and 16 wk of dietary treatment, animals fed a proatherogenic diet (CH and CF groups) had up to a 15-fold increase in plasma cholesterol levels compared with the RG and FX groups (Fig. 1A). The cholesterol levels exhibited by these cholesterol-fed rabbits are similar to those found in hypercholesterolemic patients. There was no significant difference in plasma cholesterol levels between the CH and CF groups at any point in the trials. Plasma triglyceride levels were also elevated in the CH group at all time points (Fig. 1B). The addition of 10% flaxseed to the cholesterol diet significantly attenuated this rise. A general decrease in plasma triglyceride levels was noted in the cholesterol-fed groups with an increasing length of feeding trial.


Figure 1
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  		Fig. 1. Plasma cholesterol (A) and triglyceride (B) concentrations in rabbits before (0 wk) and following 6, 8, or 16 wk of dietary interventions. RG, regular diet; FX, 10% flaxseed-supplemented diet; CH, 0.5% cholesterol-supplemented diet; CF, 0.5% cholesterol- and 10% flaxseed-supplemented diet. Values are means ± SE; n = 4–8. *P < 0.05 vs. RG and FX groups. {dagger}P < 0.05 vs. CH group.

 
Plasma total fatty acid (TFA) levels were measured in all of the groups (Table 1). The TFA content was elevated in the plasma of the cholesterol-fed groups following all end points. Notable differences in plasma fatty acid content are as follows: palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2 n-6), and arachadonic acid (C20:4 n-6) levels were all significantly higher in the CH and CF groups vs. the RG and FX groups following 6, 8, and 16 wk, with lower levels of the fatty acids in the CF group compared with the CH group at 16 wk. ALA (C18:3 n-3) levels were elevated in animals fed both flaxseed and cholesterol following all feeding durations. A small rise was noted in plasma ALA levels of the cholesterol-fed animals following 16 wk. The long-chain omega-3 fatty acid eicosapentaenoic acid (EPA; C20:5 n-3) was only detected in the cholesterol-fed groups following 16 wk, with greater levels observed in the CF group. Docosahexaenoic acid (DHA; C22:6 n-3) levels in the plasma were only detected in trace amounts. The ratios of n-6 to n-3 PUFAs were significantly lower in the flaxseed-supplemented groups (as much as 91-fold) compared with the RG and CH groups, with the difference diminishing with the length of feeding trial.


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  		Table 1. Plasma fatty acid concentrations of rabbits fed experimental diets for 6, 8, and 16 wk

 
The dietary interventions also had an effect on the levels of lipid found in the vascular tissue following 8 and 16 wk of treatment (Table 2). The TFA content was elevated in the aortic tissue of the cholesterol-fed groups, with the highest aortic TFA levels in the CF group following 16 wk. Notable differences in the aortic fatty acid content are as follows: levels of the longer-chain fatty acids (C20:1, C20:2 n-6, C20:3 n-6, C22:1, and C24:1) were elevated in the cholesterol-fed groups. Linoleic acid levels were elevated in the cholesterol-fed groups at 8 and 16 wk and were highest in the CF group at 16 wk. The long-chain PUFA arachidonic acid decreased significantly in the FX, CH, and CF groups vs. the RG group at 16 wk. Aortic ALA levels were elevated with flaxseed supplementation as well as in cholesterol-fed animals at 8 wk; however, by 16 wk, ALA levels were elevated only in the flaxseed-fed groups, with the highest levels seen in the CF group. The long-chain omega-3 fatty acid EPA was detected only in the aortic tissue of the cholesterol-fed groups at 8 wk and, at 16 wk, was only observed in the CF group. DHA levels were also detected in the FX, CH, and CF groups following 8 wk but only in the CF group following 16 wk of feeding. At both time points, the highest values were detected in the CF group. The addition of dietary flaxseed diminished the n-6/n-3 PUFA ratio in the aortic tissues of the FX and CF groups compared with the RG and CH groups.


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  		Table 2. Concentrations of fatty acids in aortic tissue from rabbits fed experimental diets for 8 and 16 wk

 
Effects of dietary interventions on atherosclerosis. Aortas were cut longitudinally, and the luminal surface was digitally photographed to measure the atherosclerotic lesion area. Plaque formation was not visible in the aortas of the control and flax-fed animals at any time point. However, extensive atherosclerotic plaques were apparent in animals consuming dietary cholesterol (Fig. 2). Both the cholesterol- and cholesterol-flax-fed groups had significantly greater plaque formation than the control and flax-fed groups following all trials. There was a statistically significant inhibition of atherosclerotic plaque formation in the cholesterol-flax group compared with the animals fed cholesterol alone at the 6- and 8-wk time points. In contrast, the CF group developed more atherosclerotic plaques than the CH group following 16 wk of hypercholesterolemic conditions.


Figure 2
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  		Fig. 2. Extent of aortic atherosclerotic lesions following 6 (A), 8 (B), and 16 wk (C) of dietary treatment. The lesion area was measured as the percentage of aortic luminal area covered by atherosclerotic lesions. Values are means ± SE; n = 3–7. *P < 0.05 vs. RG and FX groups. {dagger}P < 0.05 vs. CH group.

 
A similar qualitative effect on plaque formation was observed in cross-sectional analysis (Fig. 3). Plaques were only present in cholesterol-fed animals and were more severe in animals that did not receive flaxseed supplementation. However, at 16 wk, the protective effect of dietary flaxseed on plaque thickness were not observed.


Figure 3
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  		Fig. 3. Representative aortic cross sections from the 4 experimental groups showing Oil Red O-stained lipid deposits. RG group, 6 wk (a); FX group, 6 wk (b). Representative pictures from the RG and FX groups at 8 and 16 wk are not shown, as atherosclerotic lesions were not apparent in the RG and FX groups at any time point. CH group: 6 (c), 8 (e), and 16 wk (g). CF group: 6 (d), 8 (f), and 16 wk (h).

 
Similar results with respect to atherosclerotic plaque formation were observed in carotid vessels, although the extent of the atherosclerosis was not as severe (Fig. 4). Carotids were not collected after the 6-wk trial. After 8 wk of dietary supplementation, atherosclerotic plaque formation in the carotids was inhibited by including flaxseed in the cholesterol diet. This protective effect was lost after 16 wk of dietary intervention.


Figure 4
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  		Fig. 4. Extent of carotid atherosclerotic lesions following 8 (A) and 16 wk (B) of dietary treatment. The lesion area was measured as the percentage of luminal area of the carotid arteries covered by atherosclerotic lesions. Values are means ± SE; n = 4–7. *P < 0.05 vs. RG and FX groups.

 
Tissue weights of the 3-mm aortic sections that were used to assess vascular response were not significantly different among the four groups following the 6- and 8-wk trials. However, aortic tissue weights were significantly greater, because of large atherosclerotic lesions, in the CH and CF groups following the 16-wk dietary intervention compared with the RG and FX groups (CH, 35.4 ± 2.3; CF, 39.0 ± 4.7; RG, 21.5 ± 0.8; and FX, 20.9 ± 1.5 mg).

Effects of dietary flaxseed and cholesterol on vascular contractile response. The response of aortic rings from animals fed the different dietary regimens was investigated first as a function of the contractile agonists. No differences in KCl-induced vasoconstriction were observed in any groups following 6 and 8 wk of dietary treatment (Fig. 5, A and B). However, aortic rings from both of the cholesterol-supplemented groups exhibited an attenuated contractile response to KCl compared with the RG and FX groups after 16 wk of dietary treatment (Fig. 5C).


Figure 5
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  		Fig. 5. Contractile response to 47 mM KCl of proximal aortic rings isolated after 6 (A), 8 (B), and 16 wk (C) of dietary treatment. Values are means ± SE; n = 5–8. *P < 0.05 vs. RG and FX groups.

 
A slight depression in the contractile response to NE was observed after 6 wk of dietary intervention, as aortic preparations from both cholesterol-supplemented groups contracted significantly less in response to 10–7 M NE than did the RG and FX groups (Fig. 6A). No difference in NE-induced contraction was observed between the groups following 8 wk of dietary treatment (Fig. 6B). However, the CH and CF groups contracted significantly less in response to a range of NE concentrations (10–6, 10–5, and 10–4 M NE) compared with the RG and FX groups after 16 wk of dietary treatment (Fig. 6C). There was also a small decline in the overall contractile response to NE observed as a function of the age of all of the animals (Fig. 6, A–C).


Figure 6
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  		Fig. 6. Contractile response to increasing doses of norepinephrine (NE) of proximal aortic rings isolated after 6 (A), 8 (B), and 16 wk (C) of dietary treatment. Values are means ± SE; n = 6–8. *P < 0.05 vs. RG and FX groups.

 
Aortic relaxation responses were also monitored after precontraction with 10–6 M NE as a function of the dietary interventions. Aortic rings from the CH group exhibited significantly less endothelium-dependent relaxation in response to higher doses of ACh (10–6 and 10–5 M) than the RG group after 6 wk of dietary interventions (Fig. 7A). Flaxseed added to the diet effectively prevented these cholesterol-induced defects. After 8 wk of cholesterol feeding, the CH group relaxed less in response to 10–6 M ACh than the RG and FX groups (Fig. 7B). Again, this was prevented by including flaxseed in the diet. Following the 16 wk trial, the CH group again demonstrated a significant defect in endothelial-dependent relaxation to ACh. The addition of flaxseed partially prevented these cholesterol-induced defects in vascular relaxation; however, the protective effect no longer achieved statistical significance at concentrations of 10–5 M ACh (Fig. 7C).


Figure 7
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  		Fig. 7. Endothelium-dependent relaxation in response to acetylcholine (ACh) in aortic rings following precontraction with 10–6 M NE at 6 (A), 8 (B), and 16 wk (C) of dietary treatment. Results are presented as percentage of tension following ACh administration after precontraction with 10–6 M NE. Values are means ± SE; n = 6–8. *P < 0.05 vs. RG group. {dagger}P < 0.05 vs. CH group. #P < 0.05 vs. FX group.

 
Endothelium-independent vasorelaxation was also investigated using SNP. The results from the 16-wk dietary intervention are shown in Fig. 8. There were no significant differences in the extent of endothelium-independent relaxation to SNP among the four groups following 16 wk of dietary intervention (Fig. 8A). Furthermore, the rate of SNP-induced relaxation was also unaltered by the choice of dietary interventions (Fig. 8B).


Figure 8
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  		Fig. 8. A: endothelium-independent relaxation in response to sodium nitroprusside (SNP) in aortic rings after precontraction with 10–6 M NE, following 16 wk of dietary treatment. Results are presented as percentage of tension following SNP administration after precontraction with 10–6 M NE. B: rate of relaxation in response to 10–6 M SNP (g tension/s). Results represent the loss of tension during the first minute after the administration of 10–6 M SNP. Values are means ± SE; n = 7–8.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Flaxseed supplementation in the diet resulted in a significant elevation of ALA levels in the plasma and aortic tissue (Tables 1 and 2). This elevation occurred in the absence or presence of additional dietary cholesterol. Although the plasma concentration of the longer-chain omega-3 fatty acid EPA was only detected in low levels with cholesterol feeding following the 16 wk trial, EPA and DHA were both detected in the aortic tissue of rabbits consuming flaxseed and cholesterol following 8 wk of feeding. The highest levels were seen in animals consuming a combination of cholesterol and flaxseed following the longer feeding trials. These results demonstrate that ALA is metabolized to a small extent to longer-chain fatty acids like EPA and DHA in the rabbit. Flaxseed supplementation also reduced the n-6 to n-3 PUFA ratio, primarily as a result of the elevated levels of circulating n-3 PUFAs. The addition of dietary flaxseed to the atherogenic diet also mitigated the cholesterol-induced rise in plasma triglyceride levels (Fig. 1B). These results are consistent with previous work from our laboratory using this dietary intervention (4).

Our findings that dietary flaxseed can inhibit the development of atherosclerotic plaques on the aortic luminal surface are consistent with previous reports (41, 52, 54, 59, 64). Because dietary flaxseed supplementation did not alter circulating cholesterol levels, either in the presence or in the absence of additional cholesterol in the diet (Fig. 1A), it is clear that its anti-atherogenic effects were not achieved through a cholesterol-lowering action. These effects are consistent with previous studies (2, 4, 24, 25, 52) but in conflict with other reports that found flaxseed supplementation to lower circulating cholesterol levels (41, 54, 56, 59). Because the anti-atherosclerotic effects of fiber are due to a cholesterol-lowering action, the high-fiber content of flaxseed, therefore, is unlikely to be responsible for the anti-atherogenic action in the present study. Alternatively, the lignan SDG in flaxseed has been shown to possess potent hypocholesterolemic and anti-atherogenic properties (52, 54, 57) and is the most likely component within flaxseed to be responsible for these beneficial effects. SDG is also a potent antioxidant (50, 51, 55). Our results extend these findings to demonstrate that the protection afforded by dietary flaxseed was also observed in an important resistance artery, the carotid (Fig. 4). Thus the anti-atherogenic effects of flaxseed on atherosclerosis may have implications for the pathogenesis of stroke as well as heart disease. Consistent with this finding, several clinical studies have shown negative correlations between plasma ALA levels with the incidence of stroke (20, 37, 67) and coronary heart disease (17, 18, 27). However, our study has also identified limits to the anti-atherogenic capacity of flaxseed. The cholesterol and flax group also developed extensive atherosclerotic lesions after prolonged periods of hypercholesterolemia (Fig. 2). The extended duration of hypercholesterolemia appeared to overwhelm the beneficial effects of flaxseed supplementation.

The present study has demonstrated that dietary cholesterol had a deleterious effect on vascular contractile function (Figs. 5 and 6). These findings are consistent with the impaired vascular response of vessels exposed to a high-cholesterol environment (9, 29–31, 69). The present results extend this to an attenuation of agonist-induced vascular contractility as well as an impairment of vascular relaxation. Because we observed defects in both KCl- and NE-induced vascular tension generation, this suggests that there is a general defect in smooth muscle function. Cholesterol-induced changes in ion transport pathways including the regulation of Ca2+ homeostasis in smooth muscle cells (SMCs) may represent the mechanism for this effect (5, 6, 10, 13, 23, 65). This may explain both the contraction and relaxation defects identified in the present study. However, three additional factors may also play a role in the depressed contractile response to KCl and NE. First, tension generation was measured as a function of tissue weight. Tissue weights of the aortic rings from the cholesterol-fed animals were higher than the non-cholesterol-fed animals because the plaque increased the vessel thickness. This increase in tissue weight would tend to artificially decrease tension/tissue weight. Second, the plaque found in the aortic rings would contain nonmuscular cell types such as fibroblasts, foam cells, and macrophages that would contribute to tissue weight but not to total tension generation. Third, atherosclerosis is known to transform the phenotype of existing SMCs from a contractile to a synthetic phenotype (1, 11, 46, 66, 73). This conversion results in a loss of contractile proteins. Synthetic SMCs are no longer transcriptionally programmed to support contractile activity, resulting in an impaired vascular response in atherosclerotic vessels.

This study is the first to describe the effects of flaxseed supplementation on vascular function in atherosclerotic vessels (Fig. 7). The protective action of dietary flaxseed on vascular response was selective. Flaxseed protected against the changes in vascular relaxation but did not protect against the contractile dysfunction induced by the elevation in circulating cholesterol. The protective effect of dietary flaxseed on vascular relaxation was only attenuated following prolonged hypercholesterolemic conditions, under which flaxseed no longer prevented atherosclerotic development. Omega-3 fatty acids have been reported previously to improve vascular function by limiting the progression of atherosclerosis and reducing endothelial activation and vascular inflammation (3, 16, 40, 45). The protective effects on relaxation identified in our study clearly involved a selective, endothelium-dependent site of action. This finding is consistent with a study reporting that a high-flaxseed diet can enhance endothelial vasorelaxant function in hypertensive rats, without improving blood pressure (71). The vascular response to SNP was not different in any of the groups, demonstrating that the endothelial-independent routes of modulating vascular relaxation were unaltered (Fig. 8). There is no intrinsic change in relaxation capacity in response to nitric oxide (NO) from SNP, rather, the lesion appears to be due to the generation of NO by ACh. Because we did not observe any beneficial effects on vascular function of the flaxseed-enriched diet on its own compared with a regular diet, this would suggest that flaxseed is not altering the intrinsic characteristics of endothelial cells but instead selectively attenuates the detrimental changes induced by cholesterol. Flaxseed did not achieve this effect by altering the circulating cholesterol levels. In contrast, dietary flaxseed did increase levels of omega-3 PUFAs in the plasma and aortic tissue. The largest changes in PUFA concentrations were in ALA levels in the tissue and plasma compartments, with only minor changes in DHA and EPA levels. Therefore, it is reasonable to hypothesize that ALA induced the protective changes observed. In support of this contention, ALA levels have been shown to lower serum markers of vascular inflammation and endothelial activation (49, 60, 61, 72, 78), reduce platelet aggregation (74), and induce changes in intracellular Ca2+ movements (4, 15, 27). Furthermore, circulating ALA levels have been positively associated with endothelium-dependent vasodilation in normocholesterolemic and hypercholesterolemic subjects (19, 63, 70).

Oxidative stress may be another important mechanistic factor in cholesterol-induced vascular contractile dysfunction (22, 28, 33, 43) and in atherosclerosis (35, 57, 75). For example, statins can improve endothelium-dependent vascular relaxation in hypercholesterolemic patients by decreasing oxidative stress (33). It is possible, therefore, that the potent antioxidant capacity of flaxseed was responsible for its beneficial action on atherosclerosis and vascular relaxation (50, 54). The lignan SDG found in flaxseed is thought to be responsible for this antioxidant action (34, 51, 54, 55, 58) and may also have contributed to its protective effect against the cholesterol-induced defects in vascular relaxation.

In summary, our data demonstrate for the first time that flaxseed can protect against atherosclerotic plaque deposition in carotid arteries and confirm its anti-atherosclerotic effects in the aorta. We have also shown for the first time that dietary flaxseed can improve endothelium-dependent vascular relaxation in the presence of a high-cholesterol diet. However, dietary flaxseed is not a panacea. If the high-cholesterol diet is prolonged in duration, flaxseed eventually loses its ability to protect against cholesterol-induced lesions. Despite this limitation, our data reinforce the hypothesis that long-term supplementation of the diet with ground flaxseed may be an effective and safe dietary strategy to limit the early and moderate stages of atherogenesis and vascular dysfunction associated with atherosclerosis. The flaxseed dosage used in this investigation is similar to that used previously in human studies (14, 42). Our results, therefore, suggest that dietary modification with flaxseed may have an important protective action in humans against vascular disease in both the heart and in stroke.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a grant from the Canadian Institutes of Health Research (CIHR), the Flax Council of Canada, and the Natural Health Products Directorate. C. M. C. Dupasquier holds a Natural Sciences and Engineering Research Council of Canada Doctoral Scholarship. B. P. Ander holds a Heart and Stroke Foundation of Canada Doctoral Research Award. J. T. Wigle holds a CIHR New Investigator Award.


   ACKNOWLEDGMENTS
 
We are grateful to Polar Foods and to Dr. Edward Keneschuk for supplying the ALA-enriched flaxseed for this study. We also thank Andrea L. Edel, J. Alejandro Austria, and Riya Ganguly for valuable technical assistance in the extraction and analysis of lipids.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. N. Pierce, St. Boniface Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (e-mail: gpierce{at}sbrc.ca)

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   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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