- Top of page
- 1 Introduction
- 2 Materials and methods
- 3 Results
- 4 Discussion
- 5 References
- Supporting Information
Cardiovascular disease (CVD) is the major cause of death and disability worldwide []. An estimated 17.3 million people died from CVD in 2008, representing 30% of all global deaths []. One in four men and one in six women will die from heart disease []. Atherosclerosis, a complex process of lipid accumulation, inflammation, and plaque formation leading to narrowing of the artery, is the primary underlying cause of CVD. While family history, male gender, and ageing are unavoidable risk factors [], atherosclerosis and CVD are largely preventable. It has been estimated that 30–40% of deaths from CVD could be avoided by improved diet and lifestyle []. Elevated circulating total and LDL cholesterol, cigarette smoking, physical inactivity, obesity, hypertension, hyperhomocysteinemia, and diabetes are major independent risk factors for vascular disease []. Alcohol abuse, high salt and low wholegrain, fish, fruit, and vegetable intake are similarly associated with CVD incidence [[1, 2]].
Elevated blood concentrations of the modified amino acid homocysteine are associated with an increased risk of CVD in the general population [[3-5]]. Individuals with severe hyperhomocysteinemia (plasma Hcy in excess of 100 μmol/L) due to inborn errors in the cystathionine beta synthase (CBS) gene have a significantly increased risk of suffering a vascular event before the age of 30 years if untreated []. Low dietary intake of the B vitamins folate, B6, and B12 similarly induce moderate plasma hyperhomocysteinemia by inhibiting intracellular methionine remethylation. Hyperhomocysteinemia adversely affects vascular function in animal models via several distinct mechanisms, including homocysteine-mediated induction of endoplasmic reticular (ER) stress, aorta smooth muscle cell (SMC) growth, plasma lipid oxidation and inflammation, and increased coagulation []. Nonetheless, there remains considerable debate whether hyperhomocysteinemia is causal for CVD []. A small number of studies indicate an independent role for B vitamins in vascular health. High blood folate has been shown in prospective human studies to be independently associated with reduced risk of coronary events [[8, 9]] and carotid intima-media thickness [] while supplementation with folic acid positively influences vascular function both in hypercholesterolemic patients with CVD and in diabetics []. Similarly, supplementing at-risk patients either with folic acid alone or together with vitamins B6 and B12 reduces blood pressure [] and carotid intima-media thickening [], independently of circulating homocysteine concentrations.
A strong relationship exists between dietary saturated fat, elevated serum cholesterol, and CHD worldwide []. Moreover, increased fat intake in rodents alters lipoprotein metabolism and adipocyte function within the perivascular fat surrounding the aorta tunica adventitia []. Previously believed to have only a structural role, aorta adventitial fat has now been shown to actively regulate vascular responsiveness and function [[14, 15]]. While changes in lipid metabolism, including hyperlipidemia, are associated with hyperhomocysteinemia in humans and in homocysteine-induced vascular pathology in rodents [[16-19]], the effect of B vitamin depletion and/or hyperhomocysteinemia on lipid metabolism directly in the aorta adventitia remains to be established.
Rodent models of atherosclerosis provide a good representation of human CVD and allow comprehensive analysis of the effect of diet on disease progression. The most commonly used genetic mouse model of atherosclerosis is deficient in ApoE, which acts as a ligand for receptors that remove lipoprotein particles []. ApoE null mice spontaneously develop atherosclerotic plaques that are morphologically comparable with human lesions []. We have recently reported that atherosclerotic plaque formation in the aorta of ApoE null mice is accelerated following a moderate nutritional folate and/or B vitamin deficiency []. Here, we report that B vitamin depletion causally alters lipoprotein and fatty acid profiles both in the liver and in the vascular adventitial tissue, resulting in a proatherogenic environment directly within the aorta.
- Top of page
- 1 Introduction
- 2 Materials and methods
- 3 Results
- 4 Discussion
- 5 References
- Supporting Information
It has been suggested that hyperhomocysteinemia, which is associated with an increased risk of human CVD, may merely reflect low intracellular B vitamin status [[8-13]]. Proatherogenic changes in lipid levels are associated with low B vitamin concentrations and hyperhomocysteinemia in humans, and in homocysteine-induced vascular pathology in rodents [[16-19]]. We have shown previously that feeding a high fat diet containing cholesterol significantly exacerbates hyperhomocysteinemia and aortic plaque formation in a mouse model of nutritional folate and/or B vitamin deficiency [].
We report here how combined B vitamin depletion, rather than folate deficiency alone, may accelerate atherosclerosis in response to a high fat intake by causing proatherogenic lipoproteins, including cholesterol, to accumulate directly in the aorta tunica adventitia.
Previously believed to have a passive structural role, the perivascular adventitial fat within the tunica adventitia is now known to regulate vascular responsiveness and remodeling via secretion of vasoactive substances [[14, 15]]. Adipocyte-derived relaxing factor (ADRF) secreted by rat vascular fat cells induces vasodilation in mesenteric arteries by inhibiting the action of vasoactive modulators []. Critically, these actions are dependent on the quantity of lipid in the tissue []. Moreover, the growth of human vascular SMCs in culture is stimulated in response to conditioned medium from rat perivascular adipose tissue isolated from animals fed a HF diet for 3 months []. Dysregulation of adipocytes within the adventitia is implicated in the pathogenesis of CVD, obesity, and diabetes []. The ability of dietary factors to influence perivascular adipose lipid deposition and tissue function is currently under investigated.
Here, we measured the abundance of key lipoproteins known to influence atherosclerosis (total cholesterol, LDL cholesterol, HDL cholesterol) in lipid extracted directly from the aorta periadventitial tissue of ApoE null mice fed either a folate deficient or a combined folate, B6- and B12-depleted diet. The most significant finding from this study is that combined B vitamin deficiency, together with a high fat diet, produces a proatherogenic lipid environment directly within the aorta adventitial lipid. Feeding a hyperlipidemic and B vitamin-depleted diet increased total cholesterol and HDL cholesterol accumulation both in the liver and in adventitial lipid. B vitamin deficiency was also associated with an increase in the proportion of saturated and a decrease in monounsaturated fatty acids in both tissues. Folate deficiency alone did not substantially alter lipid metabolism. Surprisingly, combined B vitamin deficiency was associated with lower total lipid levels both in aorta and liver of ApoE mice fed a high fat diet. Hyperhomocysteinemia, while strongly linked with an increased risk of CVD, is not associated with lipid accumulation in the vasculature. Patients with severe hyperhomocysteinemia due to a mutation in the CBS gene typically do not present with conventional lipid-dense atherosclerotic plaques []. Similarly, hyperhomocysteinemia is associated with lipid-poor fibrous aortic plaques in patients with severe CVD postmortem [].
The findings from this study suggest that atherogenesis is dependent on the type of lipid that accumulates rather than on total lipid levels.
Feeding methionine in excess induces hyperhomocysteinemia in rodents and does appear to increase hepatic lipogenesis and, in some cases, plaque formation. However, the data are highly conflicting. Total cholesterol increases twofold after feeding ApoE null mice a Westernized diet []. Plasma homocysteine increased two- to threefold after methionine was added to the diet, but circulating lipid levels and plaque formation were unaltered []. Feeding methionine concurrently with an atherogenic diet containing high fat, cholesterol, and cholic acid (that increases circulating LDL and VLDL cholesterol and depresses TG) accelerates atherosclerotic plaque formation in C57Bl/6J mice but without further altering the lipid profile []. Surprisingly, feeding ApoE null mice folic acid (75 μg/kg/day for 10 weeks) on a normal chow diet background increased total plasma cholesterol (as VLDL and LDL cholesterol), yet reduced atherosclerotic plaque formation []. Homocysteine levels were unchanged in this study.
Few studies have examined mechanistically B vitamin and/or homocysteine-mediate changes in lipid metabolism and atherosclerosis. ER stress/protein unfolding or altered methylation capacity may partly explain how B vitamin deficiency and/or hyperhomocysteinemia cause lipid metabolism dysregulation. Exposing human aorta smooth muscle and liver cells in vitro to high homocysteine (1–5 mM for 48 h) increases expression of SREBP-1, a transcription factor that activates enzymes in the cholesterol/TG biosynthetic and uptake pathways []. Hyperhomocysteinemia in this system caused cholesterol accumulation intracellularly []. Similar findings have been reported in mice fed a hyperhomocysteinemic diet, with increased hepatic lipogenesis (rather than decreased lipoprotein export) causing hepatic cholesterol []. Critically, cholesterol is elevated and atherosclerosis accelerated in the aorta of hyperhomocysteinemic CBS and ApoE double knockout mice without dietary manipulation [].
Phosphatidylcholine (PC) synthesis takes place in the liver by two pathways that are both dependent on the methionine cycle. The first liver-specific pathway involves methylation of phosphatidylethanolamine (PE) to PC by phosphatidylethanolamine N-methyltransferase (PEMT) and is directly dependent on SAM availability. PC is synthesized in the second pathway from choline obtained from the diet or via the PEMT pathway [[19, 32]]. In our study, B vitamin deficiency significantly decreased hepatic SAM. This effect was aggravated by feeding a high fat diet. Reduced methylation capacity has been shown to impact on lipid metabolism in a small number of rodent studies. Liver SAM is depleted, PC is reduced and PEMT activity is inhibited in hyperhomocysteinemic CBS ± mice fed a low folate/high methionine diet []. Crucially, low folate, low SAM, and elevated homocysteine are associated with lipid deposition in the aorta of mice deficient in the folate metabolizing enzyme, MTHFR []. In addition to low intracellular SAM directly deregulating lipid metabolism, inadequate provision of SAM may also cause vascular dysfunction by causing aberrant DNA methylation and gene expression [[34-36]]. Low SAM is associated with methylation silencing and underexpression of Fads2 in CBS ± mice. The enzyme encoded by this gene catalyses metabolism of the long chain fatty acids, linoleic, and linolenic acid to arachidonic and decosahexanoic acid, respectively []. Hepatic fatty acid distribution was abnormal in these animals [].
Finally, we investigated whether induction of a proatherogenic environment in the aorta of ApoE null mice would affect circulating markers of inflammation and vascular dysfunction. Vascular inflammation and atherosclerotic plaque formation are characterized by the release of inflammatory cytokines. However, B vitamin deficiency did not change the circulating levels of any of the inflammatory markers measured, irrespective of fat intake. It remains to be investigated whether B vitamin deficiency can modify an immune response locally, possibly by affecting recruitment and/or cytokine secretion by immunologically active cells directly in the aorta tissue.
In summary, low B vitamin status, hyperhomocysteinemia, and hyperlipidemia all contribute to human CVD risk. A high fat and low B vitamin containing diet, as typically consumed in developed countries, provides a potentially considerable proatherogenic dietary stress. Here, we report that nutritional B vitamin deficiency, together with a high fat diet, promotes atherosclerosis by perturbing lipid metabolism and causing accumulation of proatherogenic lipoproteins (such as cholesterol) in the aorta. B vitamin deficiency was also associated with an increase in the proportion of saturated-free fatty acids and a decrease in monounsaturated fatty acids in this tissue. While these data generated using a mouse model of atherosclerosis must be interpreted carefully the evidence that B vitamin deficiency significantly impacts on lipoprotein deposition directly in the aorta tunica adventitia is strong.