Cardiovascular disease is the leading cause of morbidity and mortality and is responsible for approximately 30% of all deaths, claiming approximately 17 million lives per year worldwide in 2012.1–3 Furthermore, many studies have firmly established the relationship between cardiovascular disease and metabolic disorders and underlying conditions such as dyslipidemia, diabetes, and hypertension.4–8

Dyslipidemia and associated atherosclerotic/cardiovascular events can present with intense inflammation and increased production of reactive oxygen/nitrogen species (ROS/RNS) from mitochondrial oxidative stress and/or the NADPH oxidase complex, which can cause oxidative modification of LDL, thus amplifying the inflammatory potential (i.e., recruitment of phagocytes and activation of the neutrophil oxidase [Nox]-2 system) and proatherogenic events. Moreover, uncontrolled dyslipidemia can have serious consequences for the cardiovascular system, resulting in morphological changes (left ventricular hypertrophy [LVH]), dysfunction, and even heart failure.9–16

Regulation of lipid metabolism is an important target for therapeutic intervention in dyslipidemic processes to prevent or reduce the risk or severity of cardiovascular disease, and appropriate intervention can have an impact on its clinical course. However, due to the high cost, prolonged use, and especially the adverse effects associated with some lipid-lowering drugs, a drug to control dyslipidemia that presents fewer side effects and a better cost/benefit ratio is highly desirable.17–19

Studies have explored other compounds with antioxidant properties in the prevention of cardiovascular disease.10,20 Over the last few decades, nitroxides have been widely investigated because of their antioxidant capabilities.20–22 Among them, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol) is a superoxide dismutase (SOD) mimetic that shows a good partition coefficient, interacts with a broad spectrum of oxidants produced in the human body, and is able to break the chain of redox reactions.21,23,24

It has been shown that Tempol has radioprotective, chemopreventive, hypoglycemic, antihypertensive, antineoplastic, and cardioprotective effects, as well as protecting against ischemia-reperfusion injury. It also prevents obesity and neurodegenerative diseases and attenuates renal dysfunction and oxidative stress-induced injury. These biological effects derive at least partially from the ability of this nitroxide to scavenge ROS/RNS, as shown in several studies in which it has been proposed that Tempol can alleviate inflammatory diseases and reduce formation of extracellular traps (ETs) through its action on these oxidants.22,23,25 However, the beneficial anti-dyslipidemic effects of Tempol and its impact on cardiovascular events remain uncertain, and it is important to understand these actions by exploring different experimental animal models to find new therapeutic options. Therefore, the aim of this study was to assess the effects of Tempol on dyslipidemia and LVH in the well-established animal model of LDLr-/- mice.

In this study, we used a well-established animal model of LDL receptor knockout (LDLr-/-) mice, whose genetic background combined with a high-fat diet (environmental factor) is likely to lead to the development of severe dyslipidemia, and hence a high risk of cardiovascular disease. During the study, LDLr-/- mice were fed a standard or a high-fat diet for 30 days. Administration of Tempol (30 mg/kg once daily for 30 days) in mice on a high-fat diet showed a strong protective effect in controlling dyslipidemia and preventing damage to heart tissue (mitigating LVH).

LDLr-/- mice on a high-fat diet treated with Tempol showed a marked decrease in TG, LDL, and VLDL levels. Among the lipoproteins, LDL has been identified as one of the most important constituents of atheroma. As expected, LDLr-/- mice fed a high-fat diet presented a marked increase in LDL levels, and Tempol treatment decreased levels of this lipoprotein. Furthermore, increased TG levels have recently been shown to be associated with low HDL cholesterol levels, and hypertriglyceridemia is also one of the ‘deadly quartet,’ along with abdominal obesity, hypertension, and glucose intolerance.7,28,29

In our study, and in agreement with Kim et al.,11 LDLr-/- mice developed dyslipidemia with increases in TG, VLDL, and LDL (on a high-fat diet), and Tempol attenuated this condition; these results are similar to those found by Kim et al., in which Tempol treatment also prevented increases in these biomarkers of dyslipidemia.

Lipid disorders are among the most important risk factors for atherosclerotic cardiovascular disease, together with other chronic degenerative diseases with a prolonged natural history such as hypertension, obesity, and diabetes.4–7 These diseases have a complex relationship with one another; lifestyle and genetic inheritance, among other factors, are common to their etiologies. Treatment of dyslipidemia has the fundamental purpose of primary and secondary prevention of coronary artery disease, cerebrovascular disease, and peripheral arterial disease, and may also lead to the regression of xanthomas and reduce the risk of acute pancreatitis.30–32

Unlike LDL, HDL functions primarily in reverse cholesterol transport, reducing the formation of atherosclerotic plaque.33 In this study, HDL and TC levels in LDLr-/- mice did not differ significantly among the five groups evaluated. By contrast, the plasma HDL levels found by Kim et al.11 demonstrated that treatment with Tempol of apoE-/- mice maintained on a high-fat diet promoted increases in HDL levels, but these differences may be due to the characteristics of the different animal models used.34

Even on a normal diet, LDLr-/- mice may slowly develop dyslipidemia and atherosclerosis over time, and this process is accelerated with a high-fat diet.34 LDLr-/- mice develop moderate hypercholesterolemia (TC ∼250 mg/dl) when fed a standard diet and severe hypercholesterolemia when fed a high-fat diet. However, no differences between the groups were found regarding TC and HDL in this study; moreover, a closer relationship between these biomarkers was found. Furthermore, LDLr-/- mice normally become obese only when fed a high-fat diet with a fat content of more than 20%; thus, body weights differed only slightly among the groups, without statistical significance (Table 1) since only up to 20% total fat was used in this study.34

Simvastatin was used in this study as a control since this cholesterol-lowering agent is widely used preventively or as an adjuvant to correct lipid metabolism in patients with a predisposition to cardiovascular disease or primary hypercholesterolemia. Simvastatin is a prodrug that acts on the enzyme HMG-CoA reductase, preventing cholesterol synthesis and decreasing LDL, VLDL and TG.19,35 This statin, at a dose of 20 mg/kg, had significant effects on lipid profile markers and LVH, and its effects on TG, LDL, VLDL, and LVH were statistically similar to those caused by 30 mg/kg Tempol.

In LVH, increases in myocyte volume, coronary artery wall thickness, capillary rarefaction and extracellular fibrosis, and changes in energy metabolism, intracellular calcium, and myocardial contractility and relaxation are normally observed.13,14 LDLr-/- mice fed a high-fat diet for approximately 14 days develop atherosclerosis and an increased predisposition to cardiac damage, including LVH, with a mean 30% increase in cardiomyocyte diameter.26 In our study, morphological changes were observed in the untreated group of LDLr-/- mice fed a high-fat diet compared with those fed a standard diet, while treatment of these mice with Tempol attenuated these alterations, preventing LVH.

Regarding the underlying mechanisms, studies have shown that superoxide (O2-) production in LDLr-/- mice fed a high-fat diet is significantly higher than in LDLr-/- mice fed standard chow.36,37 Moreover, oxidative stress in these mice decreases nitric oxide (NO) and increases LDL oxidation. Thus, it is possible that antioxidant treatment reduces cardiac oxidative stress and prevents LVH by mitigating pathogenic mechanisms such as oxidative stress-mediated fibrosis.38

Ulasova et al. showed that quercetin, an antioxidant, can prevent LVH in ApoE-/- hypercholesterolemic mice,39 and other studies have shown that Tempol treatment can inhibit hypertension-induced oxidative stress-related LVH. In addition, Tempol (3 mmol) added to the drinking water of an experimental model of Dahl salt-sensitive rats for 10 weeks normalized LVH and reduced the cardiac expression of p22phox and Nox-2, mitochondrial uncoupling protein 2 and related oxidative stress. Thus, inhibition of cardiac ROS by Tempol prevented the cardiac fibrosis, remodeling and defective relaxation that underlie diastolic heart failure.40–42

The accumulated evidence suggests that Tempol can exert positive effects on dyslipidemia and prevent cardiac damage through multiple mechanisms; furthermore, a pleiotropic action is more plausible than a single mechanism in explaining the restoration of NO and nitric oxide synthetase (NOS) (NO is an important regulator of cardiac remodeling and is recognized as an anti-hypertrophic mediator).43,44

Furthermore, in this context, the action of Tempol in decreasing ROS is also relevant, since an increase in ROS in cardiomyocytes can activate the MAPK pathway, which has an important role in cardiac hypertrophy, and redox imbalance can also decrease the bioavailability of NO.43 Other ROS/RNS scavengers have been described as possible options for controlling atherosclerotic events and/or cardiovascular disease-related oxidative stress, since decreased antioxidant (i.e. SOD) activity and increased ROS generation have been described in such conditions, as in LVH.45–50

Several studies using other experimental models have reported the effects of Tempol on dyslipidemia, atherosclerotic events, and the cardiovascular system. The cardioprotective effect of Tempol demonstrated in this study using an LDLr-/- mice model is consistent with the findings of Zhu et al.,51 who demonstrated the action of Tempol (500 μM) on O2- in rat aortas, with a protective effect on the cardiovascular system by increasing the release of NO in the endothelium and causing vascular relaxation in aortas. Furthermore, the authors demonstrated that chronic treatment with Tempol (1 mM) added to the mice's drinking water restored the release of NO and aortic relaxation in rats.

Similarly to our findings, in an experimental model of rats with caerulein-induced pancreatitis, Marciniak et al.32 demonstrated that Tempol significantly decreased myocardial damage, mainly by attenuating oxidative stress-induced damage. Furthermore, as cited above, Kim et al.,11 using an experimental ApoE-/- mouse model, showed that Tempol (10 mg/g added to feed) can improve lipid profile, reduce the formation of pro-inflammatory cytokines and markers such as monocyte chemotactic protein and myeloperoxidase, helping reduce atherosclerotic plaque area and risk of myocardial damage.

Our study has potential limitations. Assessment of oxidative damage by measuring oxidative stress indices would have helped to demonstrate the role of oxidants in the events described herein. Measurement of other biochemical parameters such as blood glucose, interleukin-6, monocyte-chemotactic protein, myeloperoxidase, and serum amyloid A could also have supplemented these findings and established a stronger link between dyslipidemia, the genesis of atherosclerotic events, and subsequent cardiovascular complications.

Regarding the Tempol dose used in this study (30 mg/kg/day), other studies have reported the use of this nitroxide in animal models at doses of approximately 30 mg/kg or concentrations from 100 to 1000 μM/kg, once daily.11,32,51 These doses and concentrations do not display in-vivo toxicity (even doses of 300 mg/kg/day or 87 μM/kg have no toxic effects in vivo),23,52 are compatible with an EC50 for inhibiting the oxidative burst in vitro (50-400 μM),53 and concentrations up to 1000 μM appear to be safe, i.e., it does not show in-vitro pro-oxidant effects,54,55 which we consider may be a significant dose-limiting side effect in the systemic use of this nitroxide.

In addition to Tempol's previously mentioned possible side effects, caution should be exercised with nitroxides and other antioxidants because of the reductive stress that can occur with excessive use of this and other antioxidants, which may have short-, medium- and long-term effects. It has been reported that antioxidants influence the immune response (acting mainly by inhibiting the oxidative burst) and the onset of chronic diseases, such as cancer and cardiovascular disease, including cardiomyopathy.56

In summary, our study shows that the nitroxide Tempol is able to improve lipid profile and attenuate LVH in LDLr-/- mice fed a high-fat diet. These data suggest that this antioxidant can be a potent ally in preventing events related to cardiovascular disease.

The authors have no conflicts of interest to declare.