Nrf2 as a Potential Mediator of Cardiovascular Risk in Metabolic Diseases

1Although reactive oxygen species (ROS) were initially presumed to cause cell damage, they are now recognized as important molecules that regulate many cell signaling and biological processes, such as induction of defense genes, activation of transcription factors, phosphorylation of kinases, and mobilization of ions in transport systems (reviewed in;; ). In the cardiovascular system, ROS generation is important to maintain endothelial and vascular smooth muscle cells (VSMCs) function, including vascular tone control, inflammation-related responses, cell growth and proliferation, modulation of extracellular matrix production, apoptosis and angiogenesis (; reviewed in ). Alterations of the balance between cellular ROS production and the capacity to rapidly detoxify reactive intermediates play an important role in the development of risk factors for cardiovascular diseases (reviewed in and ). Such events are observed in patients with essential hypertension and various experimental models of arterial hypertension, such as spontaneously hypertensive rats (SHR) that exhibit increased levels of vascular superoxide anion and vascular smooth muscle hypercontractility. The same occurs in obesity and experimental models of diabetes, in which insulin resistance and hyperglycemia culminate in increased NAD(P)H oxidase enzyme activity and endothelial nitric oxide synthase (eNOS) uncoupling, thereby contributing to increased ROS generation and impaired vascular function.

2Vascular cells generate ROS in response to several stimuli, including cytokines, angiotensin II, endothelin-1, aldosterone and platelet-derived growth factor (PDGF) (reviewed in ). ROS signaling in endothelial cells and VSMCs involves alterations in the intracellular redox state and oxidative modification of regulatory and contractile proteins (da ). The oxidative modification of these redox-sensitive proteins alters their conformation, stability, activity and/or ability to interact with other proteins, resulting in modulation of vascular function. Redox-sensitive proteins include proteins involved in calcium handling as well as contractile proteins, proteins involved in various signaling and transcriptional activities. Redox modulation of calcium handling proteins directly affects cardiac and vascular contraction by altering intracellular calcium levels. Examples of redox-sensitive calcium handling proteins are calcium-calmodulin kinase II (CaMKII), the ryanodine receptor on the sarcoplasmic reticulum, sarcoplasmic reticulum ATPase (SERCA), and phospholamban (reviewed in and ). Protein kinases and phosphatases are also affected by direct redox modification, resulting in modulation of various signal transduction pathways in cardiac and vascular cells, including altered modulation of calcium sensitivity, phosphorylation of the myofilaments and receptor tyrosine kinase signaling (reviewed in and ).

3The long-term effects of ROS in cardiovascular function depend on the balance between signals promoting proliferation or growth inhibition and/or cell death. This dual role is evidenced by the findings that endothelial apoptosis initiated by tumor necrosis factor-alpha (TNF-α) is attenuated by ROS scavenging and that, during ischemia-reperfusion injury, ROS trigger apoptosis, whereas ROS generated during ischemic preconditioning prevent apoptosis (;; reviewed in ). In vascular cells, ROS can alter this balance leading to either excessive angiogenesis or loss of endothelial cells (reviewed in ).

4To contain ROS generation, there are antioxidant defenses that prevent or neutralize the generation of highly reactive compounds in biological systems. Among the major intracellular antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase are major players. In cooperative action, these enzymes catalyze ROS in molecular oxygen and water (reviewed in ). Other agents, such as ascorbic acid (reviewed in ) and phenolic acids (reviewed in ), which do not present enlightened mechanisms of action, also contribute to redox balance.

5Although the exact mechanisms underlying cardiovascular oxidative stress during obesity, diabetes, and atherosclerosis still remain unclear, the discovery of mechanisms and subsequent new pharmacological interventions related to the disruption of the antioxidant system has become of great interest to identify key mediators that will potentially instigate and exacerbate these cardiovascular complications.

6Several cell types throughout evolution have developed adaptive programs to counteract oxidative stress, and the role of the nuclear factor erythroid 2-related factor-2 (Nrf2) in these programs has been a subject of recent interest. Nrf2 is a basic leucine zipper region (bZip) transcription factor of the Cap n’Collar family, with approximately 589 amino acid residues and seven conserved domains (Neh1 – Neh7) (reviewed in ). When the levels of ROS and electrophiles become greater than the cell’s ability to detoxify them, a series of events results in Nrf2 activation. Activated Nrf2 heterodimerizes with Jun and small musculoaponeurotic fibrosarcoma (sMaf) proteins, translocates to the nucleus and binds to the antioxidant response element (ARE) or the electrophile-response element (EpRE) in the promoter region of Nrf2 target genes. This results in the coordinated expression and activation of antioxidant, antiapoptotic, metabolic, and detoxification proteins. Among the proteins with antioxidant activity regulated by Nrf2 are superoxide dismutase (SOD), catalase (CAT), heme-oxygenase 1 (HO-1), glutathione peroxidase 1 (GPx-1) and NAD(P)H: quinone oxidoreductase 1 (reviewed in ).

7The conserved Nrf2 domains are crucial for their self-regulation and activity. In this context, the Neh-1 domain is responsible for the binding of Nrf2 to DNA through dimerization with the low molecular weight sMaf proteins of type F, G, and K (sMafF, sMafG, and sMafK), favored by the bZip domain, and by recognition of the ARE (;; reviewed in ). The Neh-2 domain, rich in lysines, is responsible for binding of Nrf2 to its cytoplasmic repressor protein Keap-1. This domain also has high and low affinity regions for Keap-1 binding, the ETGE and DLG motifs, respectively. The Neh-3 domain, located in the carboxy-terminal portion of Nrf2, is sumoylated. This post-translational modification is important for Nrf2 transactivation. In addition to Neh-3, the Neh-4, and Neh-5 domains, when acetylated or oxidized, in oxidative stress conditions, are responsible for the transactivation of Nrf2. The Neh-6 and Neh-7 domains are responsible for the degradation of Nrf2, independently of the regulation by Keap-1.

8In physiological conditions, when there is a balance between oxidant species and the adequate activity of antioxidant proteins, Nrf2 is suppressed in the cytoplasm. This suppression occurs adjacent to the cellular cytoskeleton by the interaction of Nrf2 with Keap-1 and Cullin3, a protein of the E3 ligase family (reviewed in ). The formation of the Nrf2-Keap-1-Cullin3 complex becomes a signal for the ubiquitination and proteasome degradation of Nrf2, maintaining its basal levels along with the genes regulated by this transcription factor (; reviewed in ).

9Under oxidative or electrophilic stress conditions, ROS promote the breakdown of the Nrf2-Keap1-Cullin3 complex, allowing Nrf2 to translocate to the nucleus and activate the expression of antioxidant proteins. The ARE is normally suppressed by a heterodimer formed by the sMaf and Bach-1 proteins, preventing Nrf2 heterodimerization and binding to the ARE (reviewed in and ). However, under conditions of oxidative stress, there is an accumulation of heme groups in the cytosol, a frequent substrate for ROS formation. In this condition, Bach-1 undergoes dissociation of sMaf proteins and migrates to the cytosol to act on the heme group metabolism, allowing Nrf2 to dimerize with the sMaf protein and initiate gene transcription (reviewed in and ).

10Among the proteins that participate in Nrf2 regulation and activity, Keap-1 and Bach-1 stand out from the others due to their direct role in redox balance and cardiovascular homeostasis. Of importance, the negative regulation of Nrf2 by Keap-1 and Bach-1 raises the possibility of new therapeutic targets to prevent oxidative stress-associated cardiac hypertrophy and vascular dysfunction.

11The mechanisms accompanying the progression of obesity and its cardiovascular comorbidities have been the focus of considerable research over the last 30 years. As the mechanistic insights clarify, it is evident that the expansion of visceral adipose tissue plays a pivotal role in the pathophysiology of obesity. Increased ROS generation by the adipocytes, which, in turn, increases expression and secretion of inflammatory adipokines, causes dramatic consequences for the regulation of energy homeostasis and vascular function (; reviewed in and ). This is a common combination associated with individual cardiovascular disease risk (; reviewed in ).

12Recent evidence shows that Nrf2 is involved in the control of energy metabolism and might be a promising therapeutic target for obesity. The underlying mechanism of Nrf2 in obesity has been investigated using various experimental approaches, including Nrf2 gene deletion, Nrf2 pharmacological activators and Nrf2 gene overexpression, but only few of these approaches were clinically tested. Specific pharmacological activators of Nrf2, including epigallocatechin 3-gallate, oltipraz, sulforaphane, curcumin and 1-[2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oyl] imidazole (CDDO-Im), induce the expression and activity of Nrf2 both in vitro and in vivo. The epigallocatechin 3-gallate-induced activation of Nrf2 in liver and adipose tissue of obese mice improves lipidemic control, decreases oxidative products generation, and reduces body mass, insulin and glucose levels. Interestingly, knockout mice for the Keap-1 protein exhibit similar features, including suppression of high-fat diet-induced obesity and decreased deposition of lipids and cholesterol in the liver. Of importance, improvement of metabolic profile is closely associated with improved cardiovascular function.

13A recent study demonstrated that the Nrf2 activator sulforaphane, during revascularization procedures in metabolically compromised individuals, has the potential to suppress the progression of intimal hyperplasia. In addition, Nrf2 activation attenuates leptin-induced proliferation of VSMCs in the diet-induced obesity scenario.

14Natural compounds are also promising elements in Nrf2 activation during obesity. As an example, observed that curcumin, a natural Nrf2 activator, suppresses oxidative stress, inflammation and hypertrophy induced by treatment of cardiac cells with free fatty acids. Similarly, high-fat diet induced oxidative stress, inflammation, fibrosis, hypertrophy and tissue remodeling are attenuated by curcumin treatment. These benefits are closely associated with increased Nrf2 expression and activity, as well as reduced ROS generation.

15Deletion of the Nrf2 gene is expected to increase ROS generation and to aggravate the phenotypes of obesity. Nrf2 knockout mice show increased ROS generation, deposition of fatty acids in the liver and increased expression of genes related to the synthesis of lipids and cholesterol. Consistent with these findings, our group showed that obesity in mice favors vascular oxidative stress by increasing the expression of the downregulatory proteins of Nrf2, Keap-1 and Bach-1.

16Nrf2 in adipose tissue function and metabolic syndrome has also been examined. determined the role of Nrf2 in the development of obesity and associated metabolic disorders, using Nrf2-knockout mice on a leptin-deficient ob/ob background, a model with an extremely positive energy balance. In obese mice, ablation of Nrf2, globally or specifically in adipocytes, reduces white adipose tissue mass. However, Nrf2 deletion results in even more severe metabolic syndrome with aggravated insulin resistance, hyperglycemia, and hypertriglyceridemia. In addition, when compared to wild-type mice, the white adipose tissue of obese mice expresses substantially higher levels of many genes related to antioxidant response, inflammation, adipogenesis, lipogenesis, glucose uptake, and lipid transport. These findings support a role for Nrf2 in regulating adipose tissue development and function, insulin sensitivity, glucose and lipid homeostasis.

17In contrast to the above-mentioned study, Nrf2 knockout mice treated with a high-fat diet tend to gain less body mass and display increased insulin sensitivity and glucose tolerance. In addition, they do not exhibit increased glucose, cholesterol, or plasma triglycerides. Of importance, the altered metabolic phenotype of Nrf2- knockout mice on high-fat diet is associated with higher expression and abundance of fibroblast growth factor 21 (FGF21), a novel hormone that regulates energy metabolism, glucose tolerance and adipose tissue expansion.

18The complex roles of Nrf2 in adipogenesis and adipose tissue functions were recently examined by adipose tissue-specific ablation of Nrf2 in mice. This condition is associated with a transient delayed increase of body weight in high-fat diet-fed mice. However, the benefit is eventually suppressed after prolonged feeding. The phenotypic changes induced by adipose tissue-specific ablation of Nrf2 also extend to the whole-body level, reducing blood glucose and altering the expression of genes involved in glucose, lipid and energy metabolism. These findings are consistent with those of previous studies using Nrf2 knockout mice and provide additional mechanistic insights into the role of Nrf2 as an important mediator of glucose, lipid and energy metabolism. To date there are no studies showing direct effects of Nrf2 deletion on cardiovascular function.

19The apparent contradictory role of Nrf2 protecting mice from obesity and insulin resistance in conditions of Nrf2 deficiency in comparison to Nrf2 pharmacological activation, may be explained by the observations that Nrf2 deficiency leads to a mild increase in the levels of ROS, which stimulate the antioxidant system in a manner similar to the Nrf2 activators (reviewed in ). Another possible explanation is that Nrf2 activators also regulate non-Nrf2 signaling pathways to modulate glucose and lipid metabolism. The same is true for Keap-1, the Nrf2 repressor protein, which may also have Nrf2-independent effects on transcription factors and, consequently, on metabolic homeostasis. Controversial results on the role of Nrf2 in obesity may be linked to differences in the pathophysiological characteristics of obesity (such as diet content or time under obesity conditions), as well as specific genetic characteristics. Table 1 summarizes the contribution of Nrf2 signaling in different obesity conditions. Further studies are required to explore this apparent discrepancy on the role of Nrf2 in obesity.

20The Nrf2/Keap1/ARE signaling pathway also represents a mechanism by which the gut microbiome activates a wide range of host signaling and homeostatic processes (reviewed in ). The intestinal microbiome and its metabolites display a pivotal role in host physiological processes including immune, metabolic, neurological, and nutritional homeostasis (reviewed in ). Many of these physiological processes are under influence of ROS generation in the gut epithelia. Alterations in the gut microbiota have recently emerged as major triggers of abnormalities in the integrity of the intestinal barrier, facilitating blood translocation of bacteria and uremic toxins, systemic inflammation and adverse outcomes in obesity and diabetes (reviewed in ). Nrf2 activation in intestinal barrier leads to upregulation of antioxidant enzymes, thereby strengthening the cell’s ability to neutralize several types of free radicals. In addition, the relationship between bacterial-dependent ROS generation and Nrf2 pathway activity was recently revealed by observations that lactobacilli-induced and Nox1-mediated generation of ROS evokes Nrf2-dependent activation of cytoprotective antioxidants genes. In fact, microbe-induced ROS generation and activation of Keap1/Nrf2/ ARE signaling may contribute to our understanding on the mechanisms involved in the genesis of obesity and diabetes, comorbidities associated with increased cardiovascular risk.

21There is abundant evidence that oxidative damage caused by free radicals contributes to the pathogenesis and progression of type 2 diabetes mellitus and its complications (reviewed in; and ). Only recently, however, has the role of the Nrf2/Keap-1/ARE pathway in the pathophysiology of this condition and the wide range of its complications, such as diabetic nephropathy and impaired cutaneous wound healing begun to be elucidated (; reviewed in; ). Furthermore, as noted in an excellent and recent review on Nrf2, this pathway is implicated in diabetic damage to the pancreas and heart. There are promising results provided by animal studies and clinical trials suggesting that activation of this pathway can delay or even reverse type 2 diabetes mellitus-associated dysfunctions.

22A consistent alteration in diabetic patients is the presence of endothelial dysfunction, which precedes the development of diabetes-associated vascular complications and may explain, in part, the increased cardiovascular risk in this condition. Endothelial dysfunction in diabetes is associated with enhanced vascular contractility, oxidative stress and vascular inflammation (; and ). The importance of Nrf2 and its downstream elements in the control of vascular function in diabetes has become increasingly apparent and is reinforced by multiple studies using many of the same agents for protection from conditions other than diabetes. Bardoxolone methyl, a synthetic small molecule activator of the Nrf2/Keap-1 pathway, improves structural and functional changes in animal models of renal disease. Its therapeutic potential was extended to clinical trials in type 2 diabetic patients with chronic kidney disease. The recent studies published by Tan and Sharma’s group demonstrated that the bardoxolone analog, dh404, at low doses, attenuates atherosclerosis in diabetic Apolipoprotein E (ApoE) knockout mice and protects against diabetes-induced endothelial dysfunction, both in vivo and in vitro. Considering that endothelial dysfunction, which is the first step in the development of vascular complications in diabetes, is accompanied by pro-oxidative and pro-inflammatory processes, the atheroprotective effects of dh404 could be mediated by improvement of the endothelial function. In agreement with these observations, increased Nrf2 activity induced, for example, by bardoxolone or sulforaphane, abrogates augmented vascular contraction and attenuates reduced vasodilation in diabetic mice.

23One proposed mechanism for the improvement of endothelial function following Nrf2 activation is the increased expression of a subunit of the BK (big potassium or large conductance, Ca2+-activated potassium) channel, the BK-β1, and its attenuated degradation, a process commonly accelerated by diabetes-induced oxidative stress. Other mechanisms include reduced systemic and vascular oxidative stress as well as increased nitric oxide (NO) bioavailability. Beyond this immediate homeostatic response, long-term consequences of Nrf2 activity have also been described as important culprits of micro-and macrovascular complications associated with diabetes. Accordingly, the literature describe functional connections between Nrf2 and signaling pathways involving nuclear factor-κB (NF-κB), p53, ERK5, mTOR46, heat shock proteins, activator protein-1 (AP-1) and Notch homolog 1, translocation-associated (Drosophila) (NOTCH1). This implies that Nrf2 modulates many cellular activities, beyond its immediate homeostatic and cytoprotective actions, influencing processes as diverse as inflammation, proliferation, apoptosis, cell differentiation, tissue regeneration and even metabolism. In fact, decreased activation of Nrf2 is observed in experimental diabetic cardiomyopathy, along with a decrease in the downstream activity of antioxidant enzymes and increased oxidative stress. In this sense, activation of the Nrf2 system attenuates vascular remodeling by decreasing proliferation, migration, and fibrotic processes. These effects are mediated by reduced metalloproteinase activity and decreased protein expression of adhesion molecules and TNF-α.

24Such widespread protective effects of Nrf2 might constitute the underlying mechanism involved in the progression of diabetes-associated complications. These results have also provided strong support for the development of new potent enhancers of Nrf2 activity for the prevention and treatment of many diseases in which both inflammatory and oxidative processes have a key pathogenic role.

25Atherosclerosis, a progressing inflammatory disease produced by many risk factors, such as diabetes, hypertension, and hyperlipidemia, is one of the major cardiovascular diseases, which, together with myocardial infarction and coronary heart disease, will account for more than 20 million deaths in 2030 (reviewed in ). Even though much is known about the mechanisms that result in the formation of atherosclerotic plaque, the processes are not entirely understood.

26During the atherogenesis process, the build-up of lipids in the arterial intima triggers several changes in the microenvironment of the arterial wall, such as the formation of fatty streaks, endothelial dysfunction, recruitment and activation of immune cells and VSMCs proliferation (reviewed in ). Recruitment and retention of inflammatory cells lead to persistent production of cytokines and ROS that contribute to the progression of atherosclerotic lesion (reviewed in ). Increased ROS induces the oxidation of low-density lipoprotein (LDL) to ox-LDL that contributes to oxidative stress and foam cell formation in the arterial wall, aggravating the atherosclerotic plaque formation (reviewed in ). In this scenario, the transcription factor Nrf2 is considered a protective signaling molecule, since it induces the expression of many antioxidant genes that may attenuate atherosclerosis progression (; reviewed in ). For instance, deficiency of GPX-1, a Nrf2 target gene, in mice increases ox-LDL-induced foam cell formation and leads to amplified proliferative activity of peritoneal macrophages, indicating that this gene is atheroprotective. Moreover, atherosclerotic lesion development and oxidative stress are accelerated in HO-1 deficient ApoE knockout mice. Deletion of HO-1 in macrophages increases lipid build-up and foam cell formation and, consequently, the production of ROS and pro-inflammatory cytokines. Cheng et al. found that HO-1 expression is increased in vulnerable plaque from patients with symptomatic carotid artery disease and this increase correlates with unstable plaque phenotype. These findings indicate that HO-1 is a major regulator of advanced atherosclerotic lesion progression. However, it is not clear whether the induction of HO-1 is a compensatory atheroprotective response, trying to reduce increased levels of ROS in the plaque, or if it contributes to increased plaque vulnerability. Therefore, more studies are necessary to understand the role of HO-1 in advanced atherosclerotic plaque stage.

27On the other hand, Nrf2 gene deletion in ApoE knockout mice decreases atherosclerotic lesions at a late stage, whereas it does not affect atherosclerotic lesions in earlier stages. These observations suggest that Nrf2 inhibition may be atheroprotective in advanced plaques. Additionally, Ishii et al. observed that oxidized lipids induce Nrf2-dependent CD36 scavenger receptor expression in macrophages, resulting in intracellular accumulation of ox-LDL. However, deletion of Nrf2 in myeloid cells of LDL receptor knockout mice (LDLr-/-) exacerbates atherosclerotic lesions and increases pro-inflammatory genes expression, indicating that Nrf2 activity in myeloid cells and macrophages modulates the pro-inflammatory vascular milieu associated with atherosclerosis. These conflicting findings demonstrate that Nrf2 may also exhibit pro-atherogenic functions, depending on atherosclerotic lesion stage or animal model.

28The accumulation of lipids into the vascular intima is related to oxidative and pro-inflammatory stress that result in endothelial cells dysfunction (reviewed in ). Pro-inflammatory cytokines contribute to monocytes recruitment into the intima by inducing expression of endothelial adhesion molecules and chemokines. Nrf2 activity reduces the inflammatory response in endothelial cells. Ox-LDL-induced expression of vascular cellular adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) is reduced by a HO-1 inducer. Furthermore, Nrf2/ARE pathway suppresses TNF-α-induced expression of redox-sensitive inflammatory genes, including monocyte chemoattractant protein (MCP)-1 and VCAM-1. These results indicate that Nrf2 activation is atheroprotective due to its antioxidant and anti-inflammatory actions that limit the deleterious effects imposed by hyperlipidemic and inflammatory processes to endothelial cells.

29Nrf2 anti-atherogenic effects have also been linked to its modulatory effects on migration and proliferation of VSMCs. PDGF-induced VSMCs migration is increased by Nrf2 depletion, and Nrf2-deficient mice exhibit higher neointimal hyperplasia, as shown in a wire injury model. Nrf2 control of neointima hyperplasia may be linked to the ability of Nrf2/Keap-1 system to regulate VSMCs apoptosis and, consequently, to inhibit neointimal hyperplasia after vascular injury. Also, increased expression of Nrf2 target genes reduces VSMCs proliferation. Moreover, Nrf2 activity is important to maintain VSMCs phenotype and the activation of Keap-1/Nrf2/NQO1 pathway attenuates VSMCs calcification circulating calciprotein particles (CPP)-induced VSMCs calcification. Taken together, these studies indicate that Nrf2 may protect against atherogenesis by decreasing VSMCs migration, proliferation, calcification and vascular remodeling.

30In recent years, microRNAs (non-coding small RNAs) were identified as key regulators in the cellular events and molecular signaling pathways involved in atherosclerosis (reviewed in ). Multiple microRNAs that participate in cholesterol homeostasis (miR-33), macrophage activation (miR-155), endothelium dysfunction (miR-146), VSMCs proliferation (miR-221), and other processes that lead to plaque progression have already been identified (reviewed in ). In this context, the Nrf2 system and microRNAs can establish regulatory loops and influence vascular responses to oxidative and inflammatory injury. Accordingly, Nrf2 and miR93 regulate endothelial glycolysis, proliferation, and quiescence by Krüppel-like Factor 2 (KLF2)- and vascular endothelial growth factor A (VEGFA)-dependent mechanisms. Moreover, oxidized palmitoyl-arachidonoyl-phosphatidylcholine (Ox-PAPC)- induced HO-1 expression is partially dependent on miR-320a in endothelial cells. However, the role of microRNAs in the Nrf2 system and their implications in atherogenesis need to be further explored.

31In conclusion, anti-oxidant and anti-inflammatory effects of Nrf2 play an essential modulatory role in the formation and progression of atherosclerotic lesions, regulating functional and structural vascular responses. However, additional studies are necessary to explain the discrepant results related to the role of Nrf2 in the different stages of plaque progression. The interactions between microRNAs and Nrf2 target genes during atherosclerosis development also deserve further investigation.

32Activation of the Nrf2-dependent antioxidant system plays an important role in cell defense against oxidative stress damage, whereas the insufficiency of the Nrf2 system is associated with multiple aspects of the genesis and progression of metabolic diseases, posing a great risk to the cardiovascular system (Figure 1). The systemic increase of Nrf2 activity by several activators may be beneficial in the treatment of metabolic diseases. In addition, selective upregulation of Nrf2 genes may represent a potential therapy in obesity, diabetes and atherosclerosis. Looking to the future, experimental research that elucidates the role of Nrf2 activation in specific tissues, such as adipose tissue, liver, pancreas and others, is important for better understanding of the multiple roles of Nrf2. Additional studies may also provide new redox balance-targeted therapy for the treatment of metabolic diseases and consequent mitigation of cardiovascular risk.

33RdC, DR, CP, JS, JA, NL, and RT equally contributed to the conception and draft of manuscript and approved its final version.

34The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.