Long-term dietary nitrate supplementation slows the progression of established atherosclerosis in ApoE−/− mice fed a high fat diet

In the present study we have demonstrated a significant attenuation in the progression of established atherosclerosis in apoE−/− mice fed a HFD supplemented with nitrate. These beneficial effects appeared more pronounced in the high dose nitrate group. This improvement was associated with increased collagen expression and decreased macrophage and lipid deposition, suggesting an increase in overall plaque stability. In addition, there were significant reductions in plaque size and circulating endothelin-1 and triglycerides following high dose nitrate supplementation. Although the high dose nitrate would be difficult to achieve through dietary changes alone, it may be possible through the use of supplements and thus represents a potential new treatment option. To our knowledge, this is the first study demonstrating a beneficial effect of chronic high dose nitrate supplementation on established atherosclerosis in the apoE−/− mouse model.

Our previous study demonstrated a beneficial effect of low (0.1 mmol/kg/day) and moderate (1 mmol/kg/day) dose nitrate in preventing the development of atherosclerosis in the apoE−/− mouse, with no additional benefit observed with the high dose nitrate [18]. In the present study, a moderate and high dose of nitrate were investigated to determine any beneficial effect after atherosclerosis is already established. Despite the lack of a beneficial effect of the high dose nitrate in our previous study [18], the high dose was included in the present study in order to determine if higher doses were required when investigating a model of established disease. Indeed, our data show that the greatest effects on plaque size and composition were observed after supplementation with high dose nitrate, suggesting that preventing or reversing the progression of established atherosclerosis may require significantly greater doses of nitrate.

As expected, the HFD progressively increased body weight compared to the normal chow diet, due to the accumulation of fat mass. Nitrate treatment, at either dose, had no effect on body weight or fat accumulation. Previous studies have shown that dietary supplementation with inorganic nitrate is associated with decreased body weight [25, 26], although in these studies, mice were supplemented with nitrate from the beginning of the HFD treatment. In this current study, nitrate was supplemented in the diet at week 12 when the mice already had significant weight gain. Previous studies have demonstrated no effect of nitrate on weight in mouse models of atherosclerosis or the metabolic syndrome [18, 27]. Significant increases were also observed in TC, LDL and TG in mice fed the HFD, confirming this animal as a model of atherosclerosis. Despite this, only TG levels were significantly decreased in mice supplemented with high dose nitrate. Consistent with our finding, decreased serum TG in eNOS-deficient mice after dietary nitrate administration have previously been reported by Carlstrom’s group [26]. Elevated TG levels are observed in atherosclerosis and are recognized as a treatment target to lower cardiovascular risk [28]. Therefore, the reduction of TG levels with nitrate treatment may indicate a novel pathway by which nitrate/nitrite may affect fat metabolism or utilization of energy. Further work to investigate the beneficial effects of chronic nitrate supplementation on TC and LDL may be required.

Endothelial dysfunction is recognised as an initial first step in the development of atherosclerosis and is characterised by reduced vascular flow responses as well as lowered circulating NO levels [29]. In this study, increased levels of serum nitrate and nitrite were observed in mice supplemented with nitrate, suggesting uptake and conversion of the nitrate from the drinking water. ET-1 is a peptide predominately produced in endothelial cells where it acts as a vasoconstrictor, pro-inflammatory factor, and platelet activator [30, 31]. As such, ET-1 and NO are natural counterparts in regard to vascular function, and an imbalance in the production of these two agents may contribute to the onset of vascular dysfunction and subsequent atherosclerosis. In the present study, the HFD contributed to a significant increase in serum ET-1 levels in apoE−/− mice, indicative of endothelial dysfunction. Supplementation with nitrate, at both moderate and high doses, significantly reduced serum ET-1 levels, suggesting a protective effect on vascular function, possibly via conversion of the nitrate to nitrite and NO.

At the early stage of atherogenesis, the adherence of inflammatory cells enriched in lipids to the damaged endothelium results in the formation of a lipid-rich core. If inflammatory conditions persist, the lipid core continues to grow. Subsequently, activated leukocytes secrete proteases to degrade the extracellular matrix, meanwhile pro-inflammatory cytokines limit the synthesis of new collagen. These changes induce a thin fibrous cap and increase the risk of plaque rupture [32]. As expected, the HFD increased plaque burden compared to mice fed the normal chow diet. Supplementation with high dose nitrate reduced the total plaque burden in the HFD fed apoE−/− mice. Further investigation of plaque composition revealed that nitrate supplementation was associated with a reduction in macrophage accumulation and lipid deposition within the plaque. This change was associated with increased SMC accumulation and collagen expression within the plaque, suggestive of an increase in plaque stability [33]. The effect of nitrate on smooth muscle cell accumulation may seem counterintuitive, as it has long been proposed that NO exerts inhibitory effects on VSMC proliferation, and that nitrite also inhibits smooth muscle proliferation in models of vascular injuries [34,35,36]. However, we speculate that the accumulation of smooth muscle within the plaque is likely to be caused by the reduced macrophage content via an indirect pathway. Indeed, it has been previously demonstrated that SMC proliferation is inhibited when co-cultured with macrophages [37], suggesting that reducing the macrophage accumulation in the plaque results in a consequent removal of the inhibitory influence on SMC proliferation. The clinical implications of this finding however, remain to be elucidated.

Leptin has previously been implicated in the development of atherosclerosis due to the presence of the leptin receptor in atherosclerotic lesions [38]. High levels of leptin have been shown to increase oxidative stress in endothelial cells [39], favour VSMC migration and proliferation [40], decrease arterial distensibility [41], and contribute to obesity-associated hypertension [42]. All these effects have been found to be inversely associated with vascular health and strongly related to the pathophysiology of atherosclerosis. A previous animal model observed lower plasma leptin levels in the nitrate-fed group compared to controls [43]. Supporting this finding, we observed significantly increased serum leptin in apoE−/− mice fed a HFD, which was attenuated following high dose nitrate supplementation.

XOR has been proposed to be a major source of reactive oxygen species (ROS) and emerging evidence has suggested that XOR mediates NO formation by reducing inorganic nitrate and nitrite [44]. Several reports demonstrate a significant elevation in XOR activity and expression in models of atherosclerosis [45] as well as within plaques isolated from human patients [46]. While XOR is highly activated in liver and intestine, human endothelial cells from the microvasculature of several tissues also have high levels of XOR activity [47]. The hypoxic environment within the plaque represents an ideal environment for nitrite reduction and in particular, provides a condition to potentiate XOR-dependent nitrite reduction. The present study showed elevated XOR expression in both the liver and aorta of mice supplemented with high dose nitrate, thereby suggesting that in atherosclerosis, nitrite/nitrate bioactivity is enhanced due to the up-regulated XOR-dependent nitrite reductase activity. Previous studies in ischaemia–reperfusion injury, have shown nitrate supplementation increased nitrite reductase activity by XOR produces NO, which may protect against further injury [48]. This protection has been attributed to the inhibition of the mitochondrial respiration that limited ROS production and improved myocardial vascularization [5]. The increased expression of XOR in the present study suggests it may play a role in the protective effects of nitrate, however further work inhibiting the XOR pathway is required.

Within the body, eNOS activity is induced by various chemical factors or mechanical stimuli, which then stimulate kinases to phosphorylate eNOS, leading to an increase or decrease in eNOS activity [49]. Previous studies have demonstrated that a variety of atherogenic stimuli suppress eNOS protein levels in cultured endothelial cells [50, 51]. A significant decrease was identified in eNOS gene expression in human aortic and coronary endothelial cells from advanced atherosclerotic lesions, but not in those of early atherosclerotic samples [52]. Consistent with our findings, invesitigations in atherosclerotic animal models demonstrated unchanged or even augmented expression of eNOS in atherosclerotic arteries, despite the presence of endothelial dysfunction [53, 54]. One recent study in human coronary atherectomy specimens revealed a higher eNOS gene expression in patients with acute coronary syndromes compared to those with stable angina [55]. These results indicate that at least in the early stage of atherosclerosis, endothelial dysfunction is not attributable to a decreased expression of eNOS. Supporting this observation, we did not find significant differences in eNOS protein expression between the treatment groups. Protein phosphorylation, a key regulator of eNOS activity, is modulated by kinases, phosphatases and protein–protein interactions. The serine/threonine kinase Akt (protein kinase B), a multifunctional serine/threonine kinase, can directly phosphorylate eNOS at the serine 1177 residue, activate the enzyme and faciliate NO production [56]. In the present study, aortic eNOS and Akt activity, as demonstrated by increased phopshorlyation of these proteins, was increased with high dose nitrate supplementation. These results suggest that long term nitrate supplementation may stimulate vascular endothelial cells to produce NO via the upregulation of eNOS activity. However, it should be noted that further studies need to be conducted to ascertain the translation of our findings to humans, as it’s highly likely that species differ in both their response to and metabolism of nitrate. While the dose used in the present study is high and unlikely to be achieved through dietary changes alone, the benefits when translating these findings to humans will also need to consider the currently recommended daily intakes of nitrate of International Food Commissions. Furthermore, while this present study was designed as a proof-of-principle study, using an animal model of atherosclerosis, future studies will need to demonstrate not only if these benefits translate but also determine a dose that is efficacious and safe. It is important to be mindful that any cardiovascular benefits need to be weighed up against the potential adverse carcinogenic effects of high-dose nitrate.

The present study has demonstrated that chronic high dose nitrate supplementation can attenuate the progression of established atherosclerosis in apoE−/− mice. Mechanistically, this appears to be mediated through a XOR-dependent reduction of nitrite to NO, as well as enhanced eNOS activation via increased Akt and eNOS phosphorylation. Importantly, these beneficial effects of nitrate have been observed after disease has already been established, which has important implications when translating our findings to humans. Dietary and supplemental approaches to increase nitrate intake, which may have effects on both the nitrate-nitrite-NO pathway and eNOS-NO pathway may have therapeutic potential to attenuate atherosclerosis. While the present study suggests a potential cardioprotective effect from long-term nitrate supplementation, further work to investigate the translational aspects of these findings, including the appropriate dose as well as any potential detrimental effects of a nitrate dose outside current recommendations, in humans is required.

留言 (0)

沒有登入
gif