High Methionine and Cholesterol Diet Abolishes Endothelial Relaxation
Objective— High plasma cholesterol or homocysteine is a risk factor for atherosclerosis. Cholesterol and methionine, the precursor of homocysteine, are rarely eaten separately. Thus, the aims of this study were to determine neointima formation, aortic reactivity, and factors involved in endothelial function in rabbits fed high dietary cholesterol, methionine, or a combination of the two for 12 weeks.
Methods and Results— Rabbit dietary groups were randomized into the following: control (Con), 0.5% cholesterol (Chol), 1% methionine (Meth), and 1% methionine+0.5% cholesterol (MethChol). Aortic reactivity was studied by isometric tension techniques, aortic volumetric analysis was determined by stereological techniques, and immunohistochemistry was used to localize endothelial and inducible NO synthases, superoxide dismutase, macrophages, and nitrotyrosine. Atherosclerosis was present in the Chol and MethChol groups. Endothelium-dependent relaxation was virtually abolished in the MethChol group compared with control. Such decrease in relaxation was not attributable to a vascular smooth muscle cell defect or to a decrease in endothelial NO synthase or superoxide dismutase content. Macrophages and inducible NO synthase immunoreactivity were present in Chol and MetChol groups.
Conclusions— The combination of high dietary cholesterol plus methionine virtually abolishes endothelium-dependent relaxation, underscoring the importance of multiple risk factors in the development of cardiovascular disease.
High plasma cholesterol1 or homocysteine2 are risk factors for cardiovascular disease. They can be brought about by genetic defects in cholesterol or homocysteine metabolism or through dietary regimens. Cholesterol and methionine, the precursor of homocysteine, are rarely ingested separately, because animal products such as eggs, meat, and milk contain both cholesterol and methionine. In fact, it is estimated that 30% of the population in the United States may have high plasma levels of homocysteine2 and more than 50% have high plasma levels of cholesterol.3 Thus, it is likely that there are individuals with high plasma levels of both risk factors.
Experimental,4,5⇓ human,6–8⇓⇓ and murine genetic knockout studies (cystathionine β-synthase-deficient mice)9,10⇓ in hypercholesterolemia and hyperhomocystinemia show an impairment in endothelial-dependent relaxation to agonist stimulation in both conduit and resistance arteries, which has been partly linked to a decrease in the bioavailability of a major endothelium-derived relaxing factor, nitric oxide (NO). Because NO can inhibit the early events of atherogenesis,11 a decrease in the bioavailability of NO is thought to promote atherogenesis. The mechanisms whereby cholesterol and homocysteine decrease NO bioavailability are not well understood; however, it is popular opinion that an excess availability of the oxygen free radical O2− (oxygen stress) can scavenge NO, thus decreasing its bioavailability.12 Furthermore, the reaction of excess O2− with excess NO, which may arise from the increased expression of inducible NO synthase (iNOS)13 from macrophages and smooth muscle cells, forms peroxynitrite (ONOO−), which can nitrate the tyrosine residue of proteins forming nitrotyrosine.14 Thus, the identification of nitrotyrosine in the aortic wall by immunohistochemistry is a marker of nitrative stress.15 This is evidenced by a study from Eberhardt et al,9 who have shown that high plasma levels of homocysteine in genetically modified mice leads to increased O2− production, nitrotyrosine formation, and endothelial dysfunction. As well, a decrease in the O2− scavenger superoxide dismutase (SOD) may also increase O2− levels.16
In the present study, we tested the hypothesis that the combination of high dietary cholesterol and methionine would exacerbate the development of atherosclerosis and endothelial dysfunction compared with either agent alone. To this end, a stereological approach was used to quantitate the volume of the intima and media within a defined anatomical site of the abdominal aorta. This approach was used to avoid sampling errors attributable to plaque inhomogeneity. In addition, endothelial and smooth muscle cell function were examined, together with the immunohistochemical localization of endothelial NO synthase (eNOS), iNOS, nitrotyrosine, SOD, and macrophages in aortic rings taken from rabbits treated with either a control diet, high dietary cholesterol, methionine, or a combination of the two.
Male New Zealand White rabbits at 3 months of age were purchased from Animal Services, Monash University, Gippsland Campus, Victoria, Australia, and were randomly allocated into 4 groups and fed their respective diet for 12 weeks (n=8/group). Group 1 (control) was fed a normal rabbit chow diet; group 2 (Chol) received a normal rabbit chow diet supplemented with 0.5% cholesterol; group 3 (Meth) received a normal rabbit chow diet supplemented with 1% methionine; and group 4 (MethChol) received a normal rabbit chow diet supplemented with 1% methionine+0.5% cholesterol. The animals were housed in individual cages and maintained at a constant temperature of approximately 21°C. Food and water was supplied ad libitum. The experiments were approved by the Monash University, Department of Anatomy Ethics Committee and were carried out according to the National Health and Medical Research Council Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (6th Edition, 1997).
For information on blood sampling and analysis, tissue collection, quantification of plaque, functional reactivity studies, immunohistochemistry, and data analysis, please see the online supplement at http://atvb.ahajournals.org.
Plasma Cholesterol and Homocysteine Levels
The total plasma homocysteine, cholesterol, cholesterol/HDL ratio, and triglyceride levels are shown in the Table. Hypercholesterolemia was present in the Chol and MethChol groups, whereas hyperhomocystinemia was only present in the Meth group. Interestingly, the combination of cholesterol plus methionine did not significantly increase plasma homocysteine levels compared with control.
Please see the online data supplement.
Atherosclerosis, Intima, and Media Volumes
Sections of abdominal aorta from the Chol and MethChol groups showed a large amount of plaque compared with control and the Meth groups. The Meth group did not show plaque formation throughout the section of abdominal aorta studied; however, intimal thickening was prevalent throughout the vessel (Figure 1). The total volume of the intima was significantly increased in the cholesterol group (7.54± 1.00 mm3, P<0.01) compared with control (1.32±0.21 mm3) and additionally increased in the combination group (8.42±1.99 mm3, P<0.001) compared with control. There was no significant difference in total intimal volume between the control and Meth groups (2.42±0.23 mm3) nor between the Chol and MethChol groups. The media volume of abdominal aortic sections showed no significant difference between groups, although there seemed to be an increase in the media volumes of Chol (13.71±1.23 mm3) and MethChol groups (11.06±0.92 mm3) compared with control (7.56± 1.06 mm3) and Meth groups (9.03±1.07 mm3).
Intima to Media Ratio
The intima to media ratio in the animals fed high dietary cholesterol was significantly increased compared with control (0.54±0.05 versus 0.18±0.02, respectively, P<0.05), and this ratio was additionally increased in the MethChol group compared with control (0.77±0.16, P<0.001). There was no difference in the intima to media ratio between the control and the Meth groups (0.27±0.01).
The maximum endothelium-dependent relaxation to acetylcholine in the control group was 70.0±5.0%, with a −logEC50 value of 7.21±0.11. In both the Chol and Meth groups, there was a decreased sensitivity to acetylcholine compared with control, as well as a trend for a reduction in the maximum relaxation evoked by acetylcholine. Strikingly, the MethChol group showed virtually no endothelium-dependent relaxation to acetylcholine compared with control, and in fact, some of the aorta showed contraction to acetylcholine rather that relaxation. Importantly, there were no differences between the groups in the magnitude of initial precontraction evoked by phenylephrine (Figure 2A). All −logEC50 and maximal responses are shown in online Table I, available at http://atvb.ahajournals.org.
Endothelium-independent aortic relaxation was assessed by constructing a concentration response curve to the NO donor sodium nitroprusside (Figure 2B). The −logEC50 values indicate that the smooth muscle cell sensitivity to the vasodilator sodium nitroprusside was not affected by any of the treatments, although there was a trend for increased sensitivity and maximum responses in the MethChol group compared with control. All −logEC50 and maximal responses are shown in online Table I.
To determine whether the dietary regimens affected smooth muscle cell sensitivity to vasoconstrictors, aortic contraction was assessed by constructing a concentration response curve to the α1-adrenergic receptor agonist phenylephrine. All −logEC50 and maximal responses are shown in online Table I. Maximal contractions to phenylephrine were not significantly different between groups, although phenylephrine was less sensitive in both the Meth and MethChol groups compared with control, indicating parallel rightward shifts of the CR curves to phenylephrine in these groups (Figure 2C). Thus, these results indicate that there is a decrease in the smooth muscle cell sensitivity to the vasoconstrictor phenylephrine.
For information on the immunolocalization of eNOS, iNOS, SOD, nitrotyrosine, and macrophages, please see the online data supplement.
The main findings of the present study are that the combination of high dietary cholesterol plus methionine virtually abolished endothelium-dependent relaxation, which was not attributable to a decrease in eNOS protein or SOD protein, and that the combination of high dietary cholesterol plus methionine did not additionally exacerbate the level of atherosclerosis compared with each dietary supplement alone.
In this study, high dietary methionine feeding for 12 weeks did not lead to atherosclerosis formation in the abdominal aorta; however, intimal thickening was pronounced throughout the vessel, which may indicate the early pathological changes in the atherosclerotic cascade. These results are in accordance with a previous study by Toborek et al,17 where aortic intimal thickening was the only pathological change in 8 of 10 rabbits fed a 0.5% methionine diet for 6 or 9 months, whereas atherosclerosis was present in the other 2 rabbits studied.
There are many studies that have reported the effect of high dietary cholesterol on the development of atherosclerosis. However, in these earlier studies, the effects of high plasma cholesterol on the development of aortic atherosclerosis, as described in this study, have been previously investigated using alternate approaches, such as the oil-red O technique.18,19⇓ However, these en face preparations of tissue do not allow for the volumetric quantification of plaque or the encroachment on the lumen, nor do they detect intimal proliferation. Other investigators have quantitated atherosclerosis formation in paraffin-embedded sections, although this method of processing causes variable degrees of tissue shrinkage and thus complicates the interpretation of the end results. The present study is novel because we chose a stereological approach to quantitate atherosclerosis along a section of the abdominal aorta between 2 anatomically defined sites, and to minimize tissue shrinkage, all tissue samples were processed and embedded in glycolmethacrylate. Because the formation of plaque is heterogenous along the artery wall, using a stereological approach, we were able to prevent either an overestimation or underestimation of plaque volume when only arterial cross-sectional measurements were made. The degree of atherosclerosis in the blood vessel wall was not significantly different between the rabbits fed the high cholesterol diet and the combined cholesterol and methionine diet, suggesting that high dietary methionine may not additionally exacerbate plaque development, at least at the doses used in the present study. However, we cannot exclude the fact that atheroprotective enzymes might be highly expressed in the aortae of the combination group, which could mask the detrimental effects of the combination diet on plaque content. Indeed, recent evidence suggests that the potent antioxidant heme oxygenase-1 is highly expressed in human and animal plaques.20 If high levels of heme oxygenase-1 are found in the plaques of the combination group, this would inhibit additional atherosclerosis formation.
In these same vessels, we examined endothelial function. As expected, there was modest endothelial dysfunction in the cholesterol-fed group as seen by the rightward shift in the concentration response curve to acetylcholine compared with control. Our findings support previous studies of hypercholesterolemia4,7⇓ on endothelial function. Likewise, a similar impairment was observed in the methionine-fed group despite unconfirmed atherosclerosis, and this is supported by other animal5 and human studies.6 In contrast to the modest endothelial impairment of each diet alone, we have shown for the first time the combination of high dietary cholesterol plus methionine administered abolished endothelium-dependent relaxation to acetylcholine in the abdominal aorta. Such dysfunction seemed to be at the endothelial cell layer rather than a direct effect on smooth muscle cell reactivity, because the response to the NO donor, sodium nitroprusside, was not impaired. As well, both high dietary methionine and the combination of high dietary methionine plus cholesterol decreased the sensitivity of the smooth muscle cells to α1-adrenergic stimulation, represented by the increase in the EC50 to phenylephrine, but not the maximal response, evoked by phenylephrine compared with control. In support of these results, a study by Aleixandre et al21 showed that smooth muscle cell contractility to phenylephrine was decreased when NO bioavailability was reduced by long-term NOS inhibition in the rat. Interestingly, aortic contractile responses to other vasoconstrictors such an angiotensin II22 and thromboxane A2 analogs23 are not significantly altered by hypercholesterolemia. The mechanisms whereby reduced NO bioavailability differentially affects vasoconstriction is not clear.
The vasoconstriction to acetylcholine observed in the abdominal aorta of the rabbits fed the combination of high dietary cholesterol plus methionine is similar to the vasoconstriction described in arteries from human subjects with coronary artery disease.8 For example, in an investigation by Ludmer et al,8 vasoconstriction rather than vasorelaxation was induced by acetylcholine in all human atherosclerotic coronary arteries examined. These studies indicate that complete endothelial dysfunction can also occur in humans.
However, it is unlikely that the decrease in endothelium-dependent relaxation seen in the abdominal aorta of animals fed high dietary cholesterol or methionine is attributable to a decrease in eNOS protein or SOD enzyme. In this study, high levels of eNOS protein were evident immunohistochemically throughout the endothelial layer of the abdominal aorta in both regions free of plaque and those complicated with plaque, including evidence of eNOS within the plaque itself. Interestingly, eNOS does seem to be abundant in the rabbit model of atherosclerosis24 but decreased in human vessels with atherosclerosis25 and in human endothelial cells incubated with oxidized LDL.26 The reason for the observed discrepancy between eNOS immunoreactivity in these human studies and those described in the rabbit remains unclear. It may relate to differences in the time course of the disease. It is possible that eNOS is present in the early stages of the disease, but in the long term (many decades of life in the human), levels of eNOS may become diminished.
In addition, there was no evidence to suggest that the decrease in endothelial function observed in the experimental groups was attributable to a decrease in SOD enzyme, because SOD immunoreactivity was intense throughout the endothelial layer and within plaques of the abdominal aorta, which has also been previously reported by others.27 Interestingly, it has been suggested that the SOD enzyme in plaques may be inactive28; however, other studies have shown an increase in aortic SOD activity after high dietary cholesterol29 or methionine.30 Moreover, Miller et al31 failed to improve aortic endothelium-dependent relaxation in hyperlipidemic rabbits when endothelial O2− levels were reduced by 130% by the gene transfer of CuZn SOD or extracellular SOD. Taken together, these studies indicate that decreased endothelium dependent-relaxation does not seem to be attributable to a lack of the SOD enzyme.
Given that we have no evidence to suggest that eNOS or SOD proteins are decreased in this model, why is aortic relaxation impaired in animals exposed to high dietary cholesterol or methionine and virtually abolished when both diets are combined? The answer to this question is no doubt complex and may relate to recent evidence suggesting that eNOS may produce O2− rather than NO in a stressful milieu.32 In addition, others have suggested that the eNOS enzyme itself may be dysfunctional,33 and additional evidence for a dysfunctional eNOS protein comes from gene transfer studies, whereby the genetic transfer of eNOS to atherosclerotic vessels significantly improved vasodilation34 whereas genetic transfer of SOD to atherosclerotic vessels had no effect.31
As previously mentioned, the reaction of excess O2− with excess NO forms peroxynitrite (ONOO−), which can nitrate the tyrosine residue of proteins forming nitrotyrosine.14 Thus, the identification of nitrotyrosine in the aortic wall by immunohistochemistry is a marker of nitrative stress.15 Because the formation of nitrotyrosine in atherosclerotic plaques is not solely attributable to the reaction of the tyrosine residue in proteins with ONOO−, but also by the reaction of tyrosyl radicals, NO·2, NO−3, NO2Cl, and HOCl with tyrosine,15 the formation of nitrotyrosine is an accurate marker of nitrative stress. Thus, nitrative stress seemed to be present in the aortae of animals fed high dietary cholesterol, methionine, or a combination of the two, which is in accordance with previous reports in humans35 but not with other subjects.36 Whether the nitrotyrosine present in the vessels in this study is attributable to excess ONOO− or other nitrative compounds remains uncertain.
In support of the results presented in this study, Eberhardt et al9 have shown that in a murine model of mild hyperhomocystinemia, the mice exhibited impaired endothelial relaxation to methacholine with no visible decrease in eNOS immunoreactivity, increased nitrotyrosine immunoreactivity, and increased O2− generation in the aorta compared with wild-type mice. Furthermore, Lentz et al10 have shown in the same animal model that low dietary folate also raises plasma homocysteine levels and this leads to endothelial dysfunction.
It has been shown that macrophages within plaques produce cytokines that can stimulate iNOS production, O2− production, and a variety of biologically active substances into their local milieu.37 Our results support this idea, in that macrophages are localized in plaques in conjunction with iNOS, eNOS, SOD, and nitrotyrosine. As well, the identification of iNOS in the atherosclerotic plaques indicates that there might be high levels of NO formed within these areas.13 Indeed, such high levels of NO can lead to the formation of nitrotyrosine, which has also been identified in these plaques. It is conceivable that these toxic, cytolytic effects of excess NO may contribute to cell death and tissue necrosis commonly observed within advanced atherosclerotic lesions.38 In this regard, the excess production of NO within atherosclerotic lesions may be deleterious rather than beneficial to the function of the vessel wall. Taken together, our results and others indicate that endothelial dysfunction observed in hyperhomocystinemia or hypercholesterolemia is a multifactorial process, and additional investigations into the mechanisms of endothelial dysfunction are warranted.
An interesting finding in this study was that the plasma homocysteine level was elevated with high dietary methionine, but when both diets were combined (in the MethChol group), the plasma homocysteine levels in these animals were less than those fed with high dietary methionine alone, and although slightly elevated compared with control, these levels were not significantly different. This may represent a novel pathway of homocysteine metabolism. Indeed, cholesterol and homocysteine metabolism may be interrelated, because in vitro studies have shown that homocysteine stimulates cholesterol production in hepatocytes39 and a study in humans has shown that plasma homocysteine levels were correlated with plasma cholesterol levels.40 Taken together with our results, it seems that in vivo in the rabbit, elevations in plasma cholesterol may be affecting homocysteine levels. Although we cannot exclude the possibility that plasma homocysteine levels may be elevated in the combination group at the early stages of dietary intervention, which then decreases at the end of the dietary regime, additional studies are needed to validate this hypothesis.
In conclusion, our results show that the combination of high dietary cholesterol plus methionine administered to rabbits for 12 weeks virtually abolished endothelial function. These results suggest that the combination of high dietary cholesterol and methionine may exacerbate the onset of atherosclerosis and underscores the importance of multiple risk factors in the progression of cardiovascular disease.
- Received April 22, 2003.
- Accepted May 20, 2003.
- ↵Solberg LA, Strong JP. Risk factors and atherosclerotic lesions: a review of autopsy studies. Arteriosclerosis. 1983; 3: 187–198.
- ↵Sempos CT, Cleeman JI, Carroll MD, Johnson CL, Bachorik PS, Gordon DJ, Burt VL, Briefel RR, Brown CD, Lippel K, et al. Prevalence of high blood cholesterol among US adults: an update based on guidelines from the second report of the National Cholesterol Education Program Adult Treatment Panel. JAMA. 1993; 269: 3009–3014.
- ↵Lang D, Kredan MB, Moat SJ, Hussain SA, Powell CA, Bellamy MF, Powers HJ, Lewis MJ. Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions. Arterioscler Thromb Vasc Biol. 2000; 20: 422–427.
- ↵Woo KS, Chook P, Lolin YI, Cheung AS, Chan LT, Sun YY, Sanderson JE, Metreweli C, Celermajer DS. Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation. 1997; 96: 2542–2544.
- ↵Casino PR, Kilcoyne CM, Quyyumi AA, Hoeg JM, Panza JA. The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients. Circulation. 1993; 88: 2541–2547.
- ↵Eberhardt RT, Forgione MA, Cap A, Leopold JA, Rudd MA, Trolliet M, Heydrick S, Stark R, Klings ES, Moldovan NI, Yaghoubi M, Goldschmidt-Clermont PJ, Farber HW, Cohen R, Loscalzo J. Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia. J Clin Invest. 2000; 106: 483–491.
- ↵Lentz SR, Erger RA, Dayal S, Maeda N, Malinow MR, Heistad DD, Faraci FM. Folate dependence of hyperhomocysteinemia and vascular dysfunction in cystathionine beta-synthase-deficient mice. Am J Physiol Heart Circ Physiol. 2000; 279: H970–H975.
- ↵Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992; 6: 3051–3064.
- ↵Reiter CD, Teng RJ, Beckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem. 2000; 275: 32460–32466.
- ↵Zanetti M, Zwacka R, Engelhardt J, Katusic Z, O’Brien T. Superoxide anions and endothelial cell proliferation in normoglycemia and hyperglycemia. Arterioscler Thromb Vasc Biol. 2001; 21: 195–200.
- ↵Kanazawa K, Kawashima S, Mikami S, Miwa Y, Hirata K, Suematsu M, Hayashi Y, Itoh H, Yokoyama M. Endothelial constitutive nitric oxide synthase protein and mRNA increased in rabbit atherosclerotic aorta despite impaired endothelium-dependent vascular relaxation. Am J Pathol. 1996; 148: 1949–1956.
- ↵Oemar BS, Tschudi MR, Godoy N, Brovkovich V, Malinski T, Luscher TF. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation. 1998; 97: 2494–2498.
- ↵Liao JK, Shin WS, Lee WY, Clark SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J Biol Chem. 1995; 270: 319–324.
- ↵Morita H, Kurihara H, Yoshida S, Saito Y, Shindo T, Oh-Hashi Y, Kurihara Y, Yazaki Y, Nagai R. Diet-induced hyperhomocystinemia exacerbates neointima formation in rat carotid arteries after balloon injury. Circulation. 2001; 103: 133–139.
- ↵Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.
- ↵Govers R, Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol. 2001; 280: F193–F206.
- ↵Darblade B, Caillaud D, Poirot M, Fouque M, Thiers JC, Rami J, Bayard F, Arnal JF. Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in atheromatous rabbit aorta. Cardiovasc Res. 2001; 50: 566–576.
- ↵Takemura R, Werb Z. Secretory products of macrophages and their physiological functions. Am J Physiol. 1984; 246: C1–C9.
- ↵Werstuck GH, Lentz SR, Dayal S, Hossain GS, Sood SK, Shi YY, Zhou J, Maeda N, Krisans SK, Malinow MR, Austin RC. Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways. J Clin Invest. 2001; 107: 1263–1273.